Purified influenza A virus N2 neuraminidase vaccine is immunogenic and non-toxic in humans.

The immunogenicity and toxicity of a purified influenza virus (N2) neuraminidase vaccine (NAV) were investigated in 88 human subjects aged 18-40, and compared to response to a conventional trivalent influenza vaccine, Fluogen (Parke-Davis). NAV doses ranged from 2.6 to 69.9 micrograms and were given intramuscularly. Serologic neuraminidase-inhibiting (NI) and neuraminidase-specific ELISA responses in this N2-primed population were roughly proportional to the dose administered. Maximal response was seen in 14-21 days and NI antibody titers persisted unabated for the 6-month post-vaccination follow-up period. All doses were well tolerated with respect to local and systemic reactions. NI tests performed with the putative (1975) priming N2 antigen demonstrated anamnestic response but did not reveal responses not already shown with the homologous (1992) antigen. Response to this purified, non-adjuvanted preparation encourages continuing investigation of the induction of infection-permissive immunity with influenza virus neuraminidase.

Safety and immunogenicity of the PFP vaccine against respiratory syncytial virus (RSV): the western blot assay aids in distinguishing immune responses of the PFP vaccine from RSV infection.

PFP-1 vaccine was evaluated in a randomized, controlled study in 47 RSV seropositive children. Trivalent inactivated influenza virus (TIV) vaccine was the control. Vaccine reactions were monitored, and bloods were obtained before vaccination, 4 weeks after vaccination, and at the end of the RSV season. Respiratory illnesses were evaluated during the outbreak. Neutralizing antibody (Nt Ab) assay to RSV, IgG ELISA to RSV proteins and a Western blot assay were performed. Acute reactions with the PFP vaccine were mild. An early RSV outbreak resulted in infection of 44.4% of the TIV recipients shortly after vaccination. In the PFP vaccine groups, the Nt Ab and ELISA assays did not distinguish between Ab rises due to natural infection versus vaccine; however, the Western blot assay characterized the post-vaccine rises. Two major Western blot profiles were produced: an infection profile (antibodies that recognized the F and G surface glycoproteins and internal proteins) and a vaccine profile (antibodies that recognized only the surface glycoproteins). The PFP vaccinees who were not infected with RSV developed ELISA and Nt Ab responses to the surface glycoproteins that were similar to the TIV vaccines with natural RSV infection. None of the children developed vaccine-enhanced disease. Thus, the PFP-1 vaccine was safe and immunogenic in RSV seropositive children even when vaccine was administered during a RSV outbreak, and the Western blot assay was useful in distinguishing Ab rises caused by RSV infection versus PFP vaccine.

Description of an adenovirus type 8 outbreak in hospitalized neonates born prematurely.

An adenovirus (Ad) type 8 outbreak was prospectively studied in a neonatal intensive care unit. Nasopharyngeal secretions were cultured weekly for viruses and clinical information was obtained daily in each infant. Eleven of 112 neonates were infected with Ad; 8 of 9 available isolates represented one variant of Ad 8. The median age at infection was 54 days and the median duration of virus shedding was 2 days. Seven of 11 infants had onset of new symptoms and/or required acute respiratory support; only 2 infants had eye disease. Maternal characteristics, race, gender, age at entry into study and respiratory distress syndrome at birth were similar for both groups. Ad-infected infants tended to have earlier gestations and lower birth weights. Ad-infected neonates stayed longer in the neonatal intensive care unit, required more days of respiratory support and were more likely to develop bronchopulmonary dysplasia. Thus an association was established between lung disease in premature neonates and Ad infection.

Evaluation of an intravenous immunoglobulin preparation for the prevention of viral infection among hospitalized low birth weight infants.

An intravenous immunoglobulin (IVIG) preparation was evaluated prospectively in hospitalized low birth weight infants for the prevention of respiratory virus infection. Premature neonates were evaluated from October 19, 1987, through July 31, 1988. Nasopharyngeal secretions were cultured weekly for viruses and clinical information was obtained daily on each infant. Ninety-one infants with birth weights between 500 and 1750 g were randomized to receive either IVIG, 500 mg/kg (46 infants), or 5% albumin-normal saline (placebo), 10 ml/kg (45 infants), between Days 3 and 7 of life, 7 days later and every 14 days thereafter for a maximum of 5 doses. Demographic and life event data during pregnancy were similar for IVIG and placebo groups. Birth weight, gestational age, gender, age at entry into the study and incidence of respiratory distress syndrome at birth were also similar in both groups of premature infants. Twenty-six viruses were isolated from 25 infants. There were 13 and 12 infections in the IVIG and placebo groups, respectively. Severity of disease, as measured by clinical factors and outcomes of virus-infected infants were no different in IVIG-treated and placebo groups. Adenoviruses and cytomegalovirus accounted for 57.7 and 23.1%, respectively, of the viral isolates. In this study the use of IVIG did not prevent or modify adenovirus and cytomegalovirus infections in premature infants.

Molecular cloning and sequence determination of the fusion protein gene of human parainfluenza virus type 1.

Undegraded mRNA transcripts were isolated from human parainfluenza virus type 1 (hPIV-1)-infected LLC-MK2 cells and their size was determined through denaturing agarose electrophoresis. The two predominantly represented mRNA species (1.65 and 1.87 kb) are similar in size to other paramyxoviral mRNAs that encode their respective glycoproteins. The cDNA transcripts corresponding to these two mRNAs were used to construct two size-restricted cDNA libraries. A cDNA clone, containing a 1.87-kb insert, was identified as encoding the hPIV-1 fusion protein by positively hybridizing with a synthetic oligonucleotide mix whose sequence was derived from the conserved sequences of other paramyxoviral F0 genes. The nucleotide sequence of the cDNA insert was determined and found to contain a single, large open reading frame encoding a putative protein of 60,795 Da consisting of 556 amino acids. Comparison of the amino acid sequence with the fusion proteins of other paramyxoviruses enabled the identification of the highly conserved amino acids of the F1 N-terminus. In addition, the positions of the hydrophobic signal and transmembrane regions, cysteine, and proline residues are all conserved. These analyses confirm that the cDNA sequence is that of the F0 protein. The 5' end of the fusion protein mRNA was determined by primer extension to lie 155 bases beyond the 5' end of the cDNA insert.

Relation of serum antibody to glycoproteins of respiratory syncytial virus with immunity to infection in children.

Immunity in relation to passively transferred maternal and naturally-induced serum antibody to the viral proteins was determined in 34 children who were followed from birth through three years of age for respiratory syncytial virus infection (RSV). Sera were tested by immunoglobulin class-specific enzyme-linked immunosorbent assay using the attachment and fusion proteins of the Long strain. The basis for immunity for maternal antibody in primary infection was assessed by a comparison of the distribution of antibody titers in a) 7 children who had an upper respiratory illness to 12 whose illness was accompanied by lower respiratory disease and of b) 13 children with an RSV-associated illness in the first 6 months of life who were age-matched as to month and approximate day of birth with 11 not infected in the same period. Infection induced immunity was evaluated by a comparison of antibody titers in 19 children who were reinfected with RSV in the year following their primary infection to 15 in whom reinfection was not documented. A statistical analysis of titers revealed that antibody to the fusion protein is an important correlate of immunity. In all three comparisons, the children with less RSV disease had significantly higher IgG anti-F titers prior to infection. No differences were observed between IgA anti-F or IgG and IgA anti-G titers.

Risk of primary infection and reinfection with respiratory syncytial virus.

Respiratory syncytial virus is the most important cause of serious lower respiratory tract infection in children. For children followed up from birth in the Houston Family Study, the infection rate was 68.8/100 children less than 12 months of age and 82.6/100 during the second year of life. Virtually all children had been infected at least once by 24 months of age, and about one half had experienced two infections. Although lower respiratory tract disease (LRD) was common (22.4/100 during year 1 and 13.0/100 during year 2), most children had only one LRD illness. The risk of reinfection was inversely related to the level of neutralizing antibodies in the serum. Reinfection illnesses were generally mild, and risk of reinfection decreased to only 33.3/100 during year 4. Studies of children with LRD and surveys of hospitalizations provide the basis for an estimate of the number of children hospitalized each year during the respiratory syncytial virus epidemics. Almost 100,000 children in the United States experience an illness of sufficient severity to require hospitalization.

Parainfluenza virus type 3: seasonality and risk of infection and reinfection in young children.

In Houston the temporal occurrence of infections with parainfluenza virus type 3 has evolved from an endemic to an epidemic pattern. Continuous virological surveillance for six years demonstrated that most infections occurred the late winter or spring after influenza virus activity. At least two-thirds of children observed in the Houston Family Study were infected with this virus in each of the first two years of life, and the risk of illness was about 30/100 children per year. After two years of age, the infection and illness rates dropped to 32 and 8 per 100 child-years, respectively. Most lower-respiratory-tract disease was associated with primary infection, and the risk for infection was greater during the second year for the smaller proportion of children who escaped infection during the first year. The risk during the first year may have been modified by passively acquired maternal antibody.

Acquisition of serum antibodies to specific viral glycoproteins of parainfluenza virus 3 in children.

A radioimmunoprecipitation assay was used to study antibody responses to parainfluenza virus 3 glycoproteins in human sera. The method was not only more sensitive than the neutralization test for the detection of antibody but also provided semiquantitative assessments of the antibody response to both glycoproteins in a single assay system. Anti-hemagglutinin-neuraminidase titers were consistently higher than anti-fusion levels in the same serum specimen. Thirteen children were monitored serologically and virologically from birth until 12 months or more after their primary infection with parainfluenza virus 3. At 1 to 3 months after infection, a significant increase in the level of antibody to the hemagglutinin-neuraminidase protein developed in 12 children; of these, 9 showed rises in the level of fusion protein. In 11 of the children, antibody titers continued to rise and the geometric mean titers to the hemagglutinin-neuraminidase protein was highest in sera collected 8 to 10 months after primary infection. Reinfection as the reason for these progressive increases in antibody levels could only be confirmed for four of the children. Three other children had reinfections after the 10-month sera were obtained; in each instance the only antibody responses were to the fusion protein.

Sporadic occurrence of zoonotic swine influenza virus infections.

Two infections by swine influenza virus, antigenically similar to A/New Jersey/76 (H1N1) virus, were detected during community epidemics with other influenza viruses. The swinelike viruses were obtained during virological surveillance of acute respiratory illnesses, and the clinical symptoms of these two patients were similar to those caused by other respiratory viruses. Both patients reported contact with swine a few days before onset of illness, but in one case it was brief. Serological studies suggested that one patient may have transmitted the virus to his roommate, but spread into the community was not indicated.

Immune responses in serum and respiratory secretions following vaccination with a live cold-recombinant (CR35) and inactivated A/USSR/77 (H1N1) influenza virus vaccine.

One hundred adult volunteers were administered inactivated vaccine (20 micrograms/0.5 cc) intramuscularly (IM) or intranasally (IN), or 10(4.7) TCID50 of a live cold-adapted vaccine (CR35) IN. Microneutralization (Nt) and radioimmunoprecipitation methods were employed to measure hemagglutinin antibody responses in sera, nasal washes, and in bronchopulmonary lavage fluids. In unprimed recipients, the relative frequency of serum antibody response and magnitude of rise was highest following the IM-inactivated vaccine (100%) and lowest after IN-live vaccine (29%). However, in individuals with pre-existing antibody, the three vaccines given were comparably immunogenic. Occurrences of secretory IgA hemagglutinin antibody in nasal washings were more frequently associated with topical administration of live or inactivated vaccine, whereas, IgG hemagglutinin antibody responses occurred with equal frequency in nasal washings in all three vaccine groups. Analysis of the hemagglutinin antibody responses in the lower respiratory tract showed that the IN-live vaccine favored the induction of secretory IgA hemagglutinin antibody and the IM-inactivated vaccine stimulated a more frequent IgG hemagglutinin antibody response.

Immunity to influenza in man.

The observations summarized in this review indicate immunity to infection with type A influenza viruses is subtype specific since little or none is conveyed to subtypes possessing immunologically distinct HA and NA proteins. However, within a subtype, a prior antigenic experience with one variant may prevent or modify illness to another. The resulting degree of subtype immunity depends on the extent of relatedness between variants. Observations with H3N2 viruses indicate that homotypic resistance to subsequent infection and illness with the same virus is potent and of relatively long duration. The long lasting durability of such immunity was indicated by the epidemiologic pattern following the reappearance of H1N1 virus. Knowledge of the duration and specificity of immunity aids considerably in assessing mechanisms that account for host resistance to influenza. Recovery from influenza virus infection must involve a variety of humoral and cell-mediated immune mechanisms, and conclusions regarding the relative importance of each one are not possible at present. To prevent infection, involved immune mechanism(s) must account for: (a) subtype specificity, (b) reduced cross-reactivity of immunity for succeeding antigenic variants, (c) a long duration of immunity, and (d) immunity at the mucosal surface. Only antibody directed toward the HA molecule presently satisfies these properties and thus should be considered the major mediator of resistance to infection. Study of naturally occurring infection is needed for determining the duration and specificity of secretory IgA in nasal and lower respiratory secretions so as to establish its relative importance as a mediator of immunity. However, the described duration, specificity, and consistent relationship to immunity suggest that IgG antibody in respiratory secretions, derived entirely or partly from serum, is the most likely mediator of resistance to natural influenza.

Characterization of human serum strain-specific antihemagglutinin antibody to A/Port Chalmers/73 (H3N2) influenza virus by radioimmunoprecipitation assays.

We performed radioimmunoprecipitation assays in which iodinated preparations of A/Port Chalmers/73 (A/PC/73) hemagglutinin were used as the test antigens and high concentrations of unlabeled A/Hong Kong/68 viral protein were used to inhibit the binding of cross-reactive antibodies to quantitate strain-specific antibody responses in postvaccination sera. Strain-specific antibodies comprised 8 to 48% (mean, 20%) of the total A/PC/73 antigen-binding capacity of the sera tested. Competition radioimmunoprecipitation assays in which disrupted preparations of purified whole virus representative of several of the H3N2 variants were used indicated that the A/PC/73 strain-specific antibody that was present after adsorption of serum by A/Hong Kong/68 antigen was capable of reacting with A/England/72 and A/Victoria/75 hemagglutinins, but generally with lower avidity than with A/PC/73 hemagglutinin. A comparison of the A/PC/73 antibody titers measured by radioimmunoprecipitation and hemagglutination inhibition tests before and after adsorption with A/Hong Kong/68 whole virus suggested that cross-reactive and strain-specific antibodies were comparable in efficiency of inhibiting viral hemagglutination. These data indicated that vaccines containing later variants within a subtype could induce antihemagglutinin antibodies of restricted specificity, but that these antibodies may not be directed against unique antigenic determinant(s).

Efficacy of purified influenza subunit vaccines and relation to the major antigenic determinants on the hemagglutinin molecule.

Inactivated whole-virus vaccine of influenza A/Scotland/74 (H3N2) virus containing 700 or 1,400 chick cell-agglutinating (CCA) units, a purified subunit vaccine of equivalent dosage, or placebo were studied in 186 adult volunteers. Placebo was least reactogenic, 1,400-CCA unit whole-virus vaccine was most reactogenic, and others were intermediate. Vaccines were equally antigenic, and delineation of antibody specificities revealed antibody cross-reacting with A/Hong Kong/68 (H3N2) virus in all sera. Antibody specific for A/Hong Kong/68 virus was found in 82% of sera and for A/Scotland/74 virus in 46%. When compared with volunteers given placebo, volunteers given 700 CCA units of subunit or whole-virus vaccine exhibited significant protection against infection with live A/Scotland/74 virus. Infections in vaccinees occurred only in those with low titers of antibody to A/Scotland/74 virus, and this antibody was of the cross-reacting type. Persons with moderate and high levels of antibody resisted infection regardless of the absence or presence of antibody specific for A/Scotland/74 virus. Purified subunit vaccines provide an alternative to whole-virus preparations in primed individuals. Efficacy of vaccines may be dependent on the nature of the antibody response.

Liposomal enhancement of the immunogenicity of adenovirus type 5 hexon and fiber vaccines.

Immunogenicity of adenovirus capsid proteins carried in liposomes was comparable to that with equivalent doses administered in Freund adjuvant, and both forms were more potent than aqueous vaccines.