Harold S. Kaye

Inactivated vaccines. 2. Laboratory indices of protection

The data from our 1968-69 influenza vaccine field trials are anlaysed and pre-challenge haemagglutinin and neuraminidase serum antibodies are evaluated as indices of protection. Prevention of flu-like disease, fever, confinement to bed, and/or seroconversion to Hong Kong was significantly related to post-vaccine A/Hong Kong/68(H3N2) haemagglutination-inhibition (HI) titres. Prevention of disease was also related, although not significantly statistically in every category, to pre-challenge A/Hong Kong/68 neuraminidase inhibition (NI) titres. The trend was the same regardless of whether the origin of the NI antibody was through A/Aichi/68 or A/Japan/62 vaccines or through pre-Hong Kong influenza infections. In summarizing the data using fever as an index of disease, the attack rate (AR) among volunteers without Hong Kong NI or HI antibody was 45%. Presence of NI antibody, in the absence of HI antibody, significantly reduced the AR to 24%. Those with both NI and HI titres experienced a still lower AR of 14%. Those with HI and NI titres both > 1: 160 ran little risk of disease, with an AR of 7%.

Protein composition of coronavirus OC 43

Abstract A human coronavirus, strain OC 43, was propagated in suckling mouse brain and purified 5000-fold with, a 90% yield. Purity of the virus was confirmed by electrophoretic, ultracentrifugal, and electron microscopic procedures. Immunodiffusion and immunoelectrophoresis tests revealed one precipitin line with normal mouse brain, three with purified virus, and four with crude virus when tested against anti-pure virus or anti-crude virus animal serums. The association of a host cell antigen with the virion was confirmed by standard HI and CF tests. Polyacrylamide gel electrophoresis of solubilized purified virus revealed a minimum of six polypeptides with apparent molecular weights of 191,000 (No. 1), 104,000 (No. 2), 60,000 (No. 3), 47,000 (No. 4), 30,000 (No. 5), and 15,000 daltons (No. 6). A seventh band was occasionally found in the 165,000-dalton region of the gels. Four polypeptides contained carbohydrate and one contained lipid. Polypeptide No. 5 comprised 26% of the total viral protein and glycopolypeptide No. 3 comprised 23%. Three other components accounted for most of the remaining protein: polypeptide No. 4 (16%), glycopolypeptide No. 6 (14%), and glycolipopolypeptide No. 1 (13%). Glycopolypeptide No. 2 was 8% of the total protein. Bromelin digestion of the viral projections (spikes) removed glycopolypeptides No. 2 and No. 6. Association of the remaining polypeptides with structural components of the virion is only tentatively postulated. The buoyant density in potassium tartrate of the bromelin-treated virus was 1.15 g/cm3 and of the intact OC 43 virion was 1.18 g/cm3. By analytical ultracentrifugation the corrected sedimentation coefficient (s 0 20w) of the OC 43 virion was determined to be 390 ± 16 S, and the apparent molecular weight (MW a ) was calculated to be 112 ± 5 × 106 daltons.

Inactivated vaccines. 1. Volunteer studies with very high doses of influenza vaccine purified by zonal ultracentrifugation

In these studies very high doses (up to 4800 CCA units in adults) of formalin inactivated influenza vaccine purified by density gradient centrifugation were given safely to more than 5000 volunteers drawn from adolescent, middle aged and elderly populations. The relative paucity of reactions, compared with those groups receiving much lower doses of formalin-inactivated vaccine produced by Sharples centrifugation, suggests that reactions are due to non-viral substances rather than to toxic properties of the viruses, and that these substances are removable. Homologous serum HI antibody responses increased with increasing vaccine dosage and there was no plateau effect at the higher dose level. In those groups studied, the appearance of neutralizing activity in post-vaccination nasal washings correlated closely with the higher vaccine doses and higher HI antibody titres. The occurrence of the ‘Hong Kong flu’ epidemic in two of the groups, including over 3000 vaccine recipients, provided a unique opportunity to study the efficacy of the various vaccines used. Only those groups receiving high doses of a Hong Kong-like antigen (A2/Aichi/2/68) enjoyed significant reduction in illness (70%). Furthermore, there was a 90% reduction in the incidence of fever in that group. These results indicate that very large doses of purified influenza vaccine can be given safely. Significant prevention and modification of disease caused by influenza via the vaccine approach is dependent on the administration of large doses of the appropriate antigen.

Antigenic Relationship between Human Coronavirus Strain DC 43 and Hemagglutinating Encephalomyelitis Virus Strain 67N of Swine: Antibody Responses in Human and Animal Sera

Abstract Hemagglutinating encephalomyelitis virus of swine (HEV) was adapted to growth in suckling mouse brain. Electron micrographs of HEV-infected suckling mouse brain, prepared by negative staining and thin-section techniques, exhibited typical morphological characteristics shared with other members of the Coronaviridae. The adaptation of HEV to suckling mouse brain facilitated serologic testing by the use of common host reagents and compatible animal systems. With hemagglutination inhibition, complement-fixation, and neutralization tests, an antigenic relationship was demonstrated between human coronavirus OC 43 and HEV in specific immune and hyperimmune animal sera. Children and adults with seroconversion to OC 43 antigen had diagnostic rises in titer of antibody to HEV antigens. Individuals with seroconversion to human coronaviruses 229E and B814 demonstrated antibody to HEV but not diagnostic rises in titer. Swine with titers of antibody to HEV had lower or no detectable titers of antibody to coronavirus OC 43. Although the prevalence and geometric mean titer of antibody to DC 43 were higher than the titer of antibody to HEV in every group of normal humans tested, significant differences in antibody response to coronavirus DC 43 and HEV were seen between populations that did or did not have possible contact with swine. The evidence suggested that antibody to HEV in humans probably represented a heterologous response to infection with coronavirus DC 43. However, a heterotypic response to unknown or uncharacterized strains of coronavirus cannot be excluded.

SEROEPIDEMIOLOGIC SURVEY OF CORONAVIRUS (STRAIN 229E) INFECTIONS IN A POPULATION OF CHILDREN

Abstract The indirect hemagglutination (IHA) test for coronavirus 229E antibodies was used for serodiagnostic and seroepidemiologic studies in a population of children. Subjects ranged in age from 5 to 19 years and lived in a home which participated in a longitudinal surveillance of respiratory illness (1960–1968). During this period 1477 respiratory illnesses were observed; 63 (4%) were associated with sero-response (fourfold or greater antibody rises) to 229E. An additional 105 sero-responses were associated with unreported or subclinical illness. The frequency of these infections was cyclical, and 229E and coronavirus OC 43 infections peaked in different years among the same population. Sero-responses occurred mainly in the fall, winter and spring quarters. Preexisting antibody was demonstrated in one-third of the children with 229E seroresponses. Clinical studies indicated that the most frequent complaints with 229E infections were sore throat, coryza and cough, and the most frequent findings were pharyngeal injection, coryza and fever.

Purification and Further Characterization of an “IBV-like” Virus (Coronavirus)

Summary Examination of crude mouse brain harvest of coronavirus strain OC 43 by ultracentrifugation, sucrose density gradients, immunodiffusion, and immunoelectrophoresis demonstrated that the antigens active in the conventional CF and HA tests were associated with the virion. No soluble antigenic components were detected. Purification of the virus was achieved by adsorption and elution from human “O” erythrocytes followed by batch calcium phosphate chromatography. Studies with the purified virus confirmed our findings with the crude mouse brain harvest. Treatment of crude and purified antigen with a limited number of disrupting substances failed to yield soluble CF or HA subunits, but a systematic investigation of other techniques for releasing viral subunits is now made possible through the simple purification procedures described in this report.

Epidemic Mycoplasma Pneumoniae Infection at a College for the Deaf.

Abstract An outbreak of Mycoplasma pneumoniae infection affected up to 110 students and employees at a college for deaf students (Gallaudet). After the introduction of M. pneumoniae onto the campus in late August 1977, the disease spread slowly among the students; the last documented case occurred in mid-December. A thorough review of chart records, mail surveys, a dormitory survey, and a three-month surveillance of the student health service provided an intensive look at the epidemiology and clinical spectrum of a mild to moderately severe disease in the epidemic setting. A matched-pair analysis demonstrated a more serious clinical illness among patients with M. pneumoniae than other patients presenting with febrile respiratory illness. Appropriate antimicrobial therapy appeared to suppress a high antibody response to M. pnuemoniae. A follow-up serosurvey one year after this outbreak demonstrated diagnostic titers to M. pneumoniae in 11 of 30 patients with confirmed infection in the original epidemic.