Immunity to influenza in ferrets

Ferrets were infected with influenza A viruses and the production of serum antibodies studied using rate-zonal ultracentrifugation techniques. Following a primary infection 19S antibody was first detected in the serum, with 7S antibody occurring later. The antibody response of ferrets after a second infection with a heterotypic influenza virus appeared to be a modified primary response but occurred later. Ferrets immunized with inactivated influenza virus vaccine after prior infection with a heterotypic influenza virus produced serum antibody to the vaccine virus; this antibody response was rapid and consisted largely of 7S antibody. A secondary antibody response was also observed following infection of ferrets previously inoculated with homologous inactivated influenza virus vaccine, although no detectable serum antibody was produced after vaccination.We wish to thank Prof. Sir Charles Stuart-Harris for his advice and criticism and Mr. M. D. Denton for his excellent technical assistance. The support of the Medical Research Council is gratefully acknowledged.     

Heterotypic immunity to influenza in ferrets.

Heterotypic immunity to influenza virus in ferrets operated against heterotypic influenza viruses but not heterologous viruses. Contrary to prior reports, the protection conferred lasted for at least 18 months. This type of immunity limited virus shedding but did not prevent infection. These results suggest that this phenomenon could play a role in determining the severity of infections caused by type A influenza viruses in humans.     

Immunity to influenza

Influenza viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Improved vaccines that can overcome the decline in immune function with aging and/or can induce broader immunity to novel pandemic strains are a high priority. To design improved vaccines for the elderly, we need to better understand the effects of age on both innate and adaptive immunity. In a murine model, we have determined that defects in antigen-presenting cell (APC) expression of pattern-recognition molecules, co-stimulatory molecules, and cytokine production may play an important role in the reduced clonal expansion of T cells in aging. The use of immunomodulators such as adjuvants may overcome some of the defects of aging immunity and may also be useful in the development of improved vaccines for avian influenza A subtypes that pose a pandemic threat. Several novel strategies including the use of ISCOM-formulated vaccines, mucosal delivery, or DNA vaccination provided cross-subtype protection that could provide an important component of immunity in the event of a pandemic.     

Immunity to influenza in Ferrets

Summary Ferrets inoculated with 300 CCA of inactivated influenza A2/Hong Kong virus vaccine did not produce serum HI antibody, and were completely susceptible to subsequent infection with live A2/Hong Kong virus. Immunization of ferrets with A2/Hong Kong vaccine in Al(OH)₃ induced low levels of serum HI antibody; these animals showed a slightly reduced febrile reaction and reduced titres of virus were recovered from nasal washings following challenge virus infection. Ferrets immunized with inactivated A2/Hong Kong vaccine in Freund's incomplete adjuvant produced relatively high titres of serum HI antibody, but did not produce local antibody detectable in nasal washings. After challenge infection, these animals showed a modified febrile reaction, lower titres of virus were recovered from nasal washings and nasal symptoms were reduced. These results, together with results of similar studies, indicated that the degree of immunity to challenge virus infection was related to the titre of serum HI antibody. However, none of the methods used to induce serum HI antibody gave as solid an immunity as found following live virus infection, although immunization could induce levels of serum HI antibody comparable to that found following virus infection.     

Immunity to influenza in ferrets. XIV: Comparative immunity following infection or immunization with live or inactivated vaccine.

Immunization by live influenza virus induced a greater protective effect against subsequent challenge by the homologous virus than by the corresponding killed virus vaccine. Furthermore, tracheas excised from 11-day and 28-day influenza-virus-infected ferrets were more resistant to reinfection than tracheas excised from ferrets immunized by killed influenza vaccine, despite equivalent serum antibody titres at these times. Histological examination of trachea sections taken from vaccinated and virus-infected animals showed an increased cellular inflammatory infiltrate in the latter at Days 11 and 28 after immunization. The amount of IgG detected in these sections, measured by a fluorescent antibody technique, correlated with the extent of cellular infiltration, the fluorescence being both intra- and extracellular for sections from virus-infected animals, but only extracellular in sections from Day-28 vaccinated animals. In contrast there was little or no cellular infiltration into lung tissues, the levels of IgG detected being comparable to those in sections taken from control animals. These results provide further evidence that live influenza vaccines induce local antibody in the upper respiratory tract of ferrets, in contrast to killed influenza vaccines, and that this local induction may play a significant role in the greater protective efficacy of live influenza vaccines.     

Local and systemic immunity to influenza infections in ferrets.

To establish whether immunity to influenza infection in the ferret is local or systemic, two sites of challenge were utilized: the nose and the anatomically isolated tracheal pouch. Infection of either site did not spread to the other site, and challenge of either site resulted in seroconversion by 13 days. Simultaneous challenge of both sites 21 days after the primary infection revealed that prior infection of the pouch prevented subsequent reinfection of the pouch, but not infection of the nose. Thus, systemic immunity did not prevent the initiation of nasal influenza infection in the ferret. However, the duration of virus shedding from the nose was reduced to half of that seen when ferrets were infected for the first time, showing that the prior pouch infection did lead to a more rapid recovery from the subsequent nasal infection. Passively administered anti-influenza antibody did not prevent or modify the nasal infection, but it did prevent the pouch infection. This is consistent with the observation that an initial infection of the nose prevented pouch infection upon challenge 21 days later. The prior nasal infection also prevented the subsequent nasal infection. These data suggest that immunity to acquisition of influenza infection in the ferret is a local phenomenon, whereas recovery from active infection is influenced by systemic immune mechanisms.     

Production of highly cross-reactive hemagglutination-inhibiting influenza antibodies in ferrets.

Ferrets were sequentially infected at time intervals of 3 weeks with different influenza virus A strains. It was found that secondary infection can result in the appearance of antibodies highly cross-reacting with a virus strain closely related to the strain of first infection. Such highly cross-reacting antibodies were designated as HCR antibodies. Evidence is presented that HCR antibodies were not antineuraminidase antibodies and, in addition, were not specifically oriented to the strain with which the crossing was observed. When using time intervals of 3 weeks between infections, no increase after secondary infection of antibodies oriented to the strain of first infection was recorded. However, when time intervals of 4 to 5 months between infections were used, secondary infections resulted in an increase of antibodies oriented to the strain of first infection ("original antigenic sin") but not in the appearance of HCR antibodies. In addition, antibodies combining specifically with both infecting strains, designated as doubly specific antibodies, were found. Thus, the conclusion was reached that the original antigenic sin phenomenon and the appearance of HCR antibodies are mutually exclusive events.     

The role of lung development in the age-related susceptibility of ferrets to influenza virus

Newborn (I-day-old) ferrets died following intranasal inoculation of influenza virus (clone 7a) but suckling (15-day-old) ferrets were almost as resistant as adult ferrets. Many of the deaths in newborn ferrets were consequent upon an increased lower respiratory tract infection. One reason for the latter was an increase in susceptibility of both ciliated epithelium and alveolar cells in newborn ferret lungs when compared with the corresponding cells in adult and suckling ferrets (Coates et al. 1984). Work reported here shows that the lungs of newborn ferrets possess a greater proportion of ciliated epithelium-lined airway in comparison with the lungs of suckling and adult ferrets. This situation might also contribute to the increased susceptibility of the lower respiratory tract although the difficulties of assessing this influence precisely are discussed. In addition, the occlusion of the narrower airways of the immature lung in the infected newborn ferret contributes to the increased respiratory complications.     

The role of lung development in the age-related susceptibility of ferrets to influenza virus.

Newborn (I-day-old) ferrets died following intranasal inoculation of influenza virus (clone 7a) but suckling (15-day-old) ferrets were almost as resistant as adult ferrets. Many of the deaths in newborn ferrets were consequent upon an increased lower respiratory tract infection. One reason for the latter was an increase in susceptibility of both ciliated epithelium and alveolar cells in newborn ferret lungs when compared with the corresponding cells in adult and suckling ferrets (Coates et al. 1984). Work reported here shows that the lungs of newborn ferrets possess a greater proportion of ciliated epithelium-lined airway in comparison with the lungs of suckling and adult ferrets. This situation might also contribute to the increased susceptibility of the lower respiratory tract although the difficulties of assessing this influence precisely are discussed. In addition, the occlusion of the narrower airways of the immature lung in the infected newborn ferret contributes to the increased respiratory complications.     

Bacterial sinusitis and otitis media following influenza virus infection in ferrets

Streptococcus pneumoniae is the leading cause of otitis media, sinusitis, and pneumonia. Many of these infections result from antecedent influenza virus infections. In this study we sought to determine whether the frequency and character of secondary pneumococcal infections differed depending on the strain of influenza virus that preceded bacterial challenge. In young ferrets infected with influenza virus and then challenged with pneumococcus, influenza viruses of any subtype increased bacterial colonization of the nasopharynx. Nine out of 10 ferrets infected with H3N2 subtype influenza A viruses developed either sinusitis or otitis media, while only 1 out of 11 ferrets infected with either an H1N1 influenza A virus or an influenza B virus did so. These data may partially explain why bacterial complication rates are higher during seasons when H3N2 viruses predominate. This animal model will be useful for further study of the mechanisms that underlie viral-bacterial synergism.     

Immunity to influenza virus infection induced by heterologous,inactivated vaccines

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.In a volunteer study, groups of students were immunized with 400 IU of the same vaccines as used above. The ability to infect these volunteers with WRL 105 virus given 4 weeks later was directly related to the vaccine-induced serum HI antibody to the challenge virus. The highest titers of serum HI antibody to A/Scotland virus were found in volunteers inoculated with homologous vaccine, lower titers were found in volunteers given A/Port Chalmers or A/England/ 72 vaccine and the lowest levels were seen in volunteers given A/Hong Kong/68 vaccine: the largest number of infections by the challenge virus was seen in volunteers given A/Hong Kong/68 vaccine, less were observed in volunteers given A/England/72 vaccine, and least were found in groups given A/Port Chalmers or A/Scotland/74 vaccine. Compared with the incidence of infection in volunteers given B/Hong Kong/73 vaccine, all groups given heterologous influenza A vaccines showed some immunity to challenge infection.     

Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.