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 X. Intranasal Immunization of Ferrets with Inactivated Influenza A Virus Vaccines

The response of ferrets after intranasal inoculation of inactivated A/Hong Kong/68 (H3N2) influenza virus vaccines is reported. Normal ferrets given either saline vaccine in drops or freeze-dried vaccine in an aerosol intranasally did not produce detectable serum or nasal hemagglutination inhibiting antibody and were found to be completely susceptible to challenge infection with A/Hong Kong/68 virus. Intranasal saline vaccine did not produce an additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant. Ferrets primed by previous infection with A/PR/8/34 (H0N1) influenza virus, however, responded to intranasal immunization with saline A/Hong Kong/68 virus vaccine and produced serum and nasal antibody. These animals were found to be partially resistant to challenge infection, in contrast to similar animals given saline vaccine intramuscularly which were completely resistant to challenge infection. Primed ferrets did not respond after immunization with the freeze-dried aerosol vaccine, but this may have been due to a failure of the aerosol to be inhaled satisfactorily.     

Immunity to Influenza in Ferrets: II. Influence of Adjuvants on Immunization

Immunization of ferrets with a single intramuscular inoculation of killed A2/Hong Kong virus did not induce serum or nasal antibody, and these animals were found to be completely susceptible to subsequent infection with virulent influenza virus A2/Hong Kong/3/68. A similar result was found for ferrets immunized with 2 inoculations of killed virus vaccine given 2 weeks apart. Ferrets immunized with killed A2/Hong Kong virus in conjunction with Bordetella pertussis produced relatively low levels of serum HI antibody to A2/Hong Kong virus; when infected with virulent influenza virus, these ferrets showed a modified reaction, with a less marked febrile reaction than was observed for non-immunized animals.Immunization of ferrets with killed A2/Hong Kong virus in Freund''s complete adjuvant resulted in the production of relatively high levels of serum HI antibody, but no detectable nasal antibody. These animals were shown to be partially immune to subsequent infection with virulent influenza virus. However, although the serum antibody levels of these animals following immunization was comparable to that found following infection with live virus, the degree of immunity to infection with virulent influenza virus was measurably less.     

Immunity to Influenza in Ferrets: I. Response to Live and Killed Virus

Ferrets were found to react with a sharp febrile response to intranasal infection with influenza virus A2/Hong Kong/3/68. Virus was recovered from nasal washings taken 3 days after infection, and virus antibody was found in serum specimens taken 21 days after virus infection. Virus infection produced a pronounced rhinitis; the protein concentration in nasal washings was found to increase three to five-fold with peak levels occurring on day 7, post-infection. Concomitant with the increased protein levels, detectable levels of HI and neutralizing antibody were found in the nasal washings. However, nasal washings taken 13 days or more after influenza virus infection did not contain either increased levels of protein or detectable antibody. These ferrets were immune to re-infection with homologous virus inoculated 5 weeks after primary infection. Thus, ferrets showed no febrile response; virus was not recovered from nasal washings; serum antibody titres did not increase; no increase in protein levels was found in nasal washings; and HI antibody was not found in nasal washings.Using these criteria to assess susceptibility or immunity to influenza virus infection, infection with attenuated influenza virus A2/Hong Kong/1/68 produced immunity to re-infection with virulent virus. Ferrets infected with influenza virus B/England/13/65 or immunized with killed A2/Hong Kong virus did not induce any immunity to infection with influenza virus A2/Hong Kong/3/68.     

The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection

The ability of several, live type A influenza viruses to enhance the serum haemagglutination-inhibiting (HI) antibody response of hamsters to subsequent immunization with inactivated, heterotypic influenza virus vaccines was examined. Live influenza viruses were found to vary in their priming ability for a given vaccine, and a given virus was not able to prime for all inactivated vaccines to an equal extent. Common determinants in the haemagglutinin antigens of the priming virus and the vaccine virus were suggested as responsible for the enhancement of the antibody response to some of the vaccines, but for other pairs of viruses the haemagglutinin antigens were distinct. Thus, enhancement in these instances cannot be due to cross-reacting haemagglutinins. Pre-infection of hamsters by several influenza type A viruses was employed in an attempt to enhance the serum HI antibody response to purified, haemagglutinin antigens prepared from A/PR/8/34 and the MRC-2 recombinant strain of A/England/42/72 viruses. Although prior infection enhanced the antibody response to whole virus, this was not demonstrable for the purified haemagglutinin components of the virus. The possible reasons for this are discussed.     

Strain specificity of the antineuraminidase antibodies of influenza virus serotype N2 strains in convalescents

Antineuraminidase antibody was determined in the subjects who had suffered influenza during the epidemics of 1970-1975 in the GDR. As early as 1970 the highest titers of antibody (greater than or equal to 1:60) were found not only to the prototype A/Hong Kong/1/68 strain but also to its subsequent drift variants A/England/42/72, A/Port Chalmers/1/73. Some subjects had antineuraminidase antibody to avian influenza virus.     

Immunological potency of a subunit influenza vaccine in adults

Summary The immunological potency of a subunit influenza vaccine (from A/England/42/72 virus) and of two commercial whole-virus vaccines (containing either A/England/42/72 or A/Hong Kong/68 virus) was studied in adults in an industrial plant. Serum samples were taken in vaccinated and control non-vaccinated subjects prior to and three weeks and five months after the vaccination. Most of the vaccinees developed high levels of hemagglutination inhibiting antibodies against all the type A influenza viruses employed in the test; these antigens included a new strain (A/Dunedin/73) that had not previously circulated in Czechoslovakia. The antibody response after the subunit vaccine was somewhat better than after the whole-virus vaccines administration. The whole-virus vaccine from the A/England/42/72 virus was more efficient in inducing antibody response against the more recent isolates than the A/Hong Kong/68 virus vaccine.     

Immunity to influenza in ferrets

Ferrets were infected with recombinant influenza A viruses which possessed either the haemagglutinin or neuraminidase antigens of A/Hong Kong/68 influenza virus. After five weeks the immunity of the animals was challenged by infection with A/HK/68 virus. Immunity to challenge infection was greatest in those ferrets with serum HI antibody to A/HK/68; the presence of NI antibody conferred a measurably lower degree of immunity. A small degree of heterotypic immunity was observed following challenge infection of ferrets previously infected with influenza virus A/PR/8/34, although the surface antigens of this virus are completely different from those of A/HK/68. Experiments in which ferrets were infected with A/HK/68 virus and subsequently challenged with the recombinant viruses confirmed the results of the first experiment.     

Failure of attenuated temperature-sensitive influenza A (H3N2) virus to induce heterologous interference in humans to parainfluenza type 1 virus.

The present investigation was undertaken to determine if a candidate live vaccine virus, influenza A/Hong Kong/68-ts-1 [E] (H3N2), induced heterologous interference against an interferon-sensitive, wild-type, parainfluenza type 1 challenge virus. The parainfluenza virus was administered 7 days after Hong Kong/68-ts-1 [E] virus infection. The clinical response, daily quantitative virus shedding, interferon production, and serum and nasal wash antibody responses were determined in an experimental group (influenza A virus followed by parainfluenza virus) and 10 volunteers in a control group (parainfluenza virus only). The volunteers were selected on the basis of susceptibility to the two viruses, i.e. serum hemagglutination-inhibition antibody titer of is less than or greater to 1:8 for influenza virus and low nasal wash antibody titer (is less than or greater to 1:8) for parainfluenza virus. Despite a 100% infection rate in the Hong Kong/68-ts-1 [E] vaccinees, no heterologous interference was induced against the parainfluenza type 1 virus challenge.     

Antibody Response of Hamsters to A2/Hong Kong Virus Vaccine after Priming by Heterotypic Virus Infection

Hamsters previously infected with influenza virus A1/FM/1/47 produced serum hemagglutination inhibition (HI) antibody in response to 1/100 the antigenic dose of inactivated influenza virus A2/Hong Kong vaccine necessary to induce antibody in normal animals. This priming effect was believed to be due to the virus infection which caused an immune response to a virus antigen common to both the infecting virus and the virus vaccine; this antigen acted as a carrier for the specific vaccine virus hemagglutinin and potentiated the immune response to the new antigen. This theory, which has been established in other immune systems, was tested, and the results obtained did not contradict the conditions imposed in the above explanation. Thus, the priming effect could be transferred to normal hamsters by inoculation of spleen cells from virus-infected animals, and the HI antibody response to the virus vaccine was characteristic of a secondary response. The theory also required that the new antigen be coupled to the carrier protein; however, primed hamsters produced serum HI antibody after inoculation with ether-Tween-split virus vaccine, but there was no proof that this vaccine was completely dissociated.     

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).     

Immunity following intranasal administration of an inactivated,freeze-dried A/England/42/72 vaccine

A group of 23 student volunteers were each inoculated intranasally with 400 IU of inactivated, freeze-dried A/England/42/72 vaccine. Only one volunteer showed a four-fold rise in serum HI antibody following immunization, and the mean increase in serum HI antibody (gmt) for all volunteers did not increase two-fold. Thirteen of the volunteers developed detectable levels of nasal wash neutralizing antibody after immunization; local antibody was most commonly found in volunteers who produced a detectable but less than four-fold fise in serum antibody titre, and who produced nasal washings with relatively high concentrations of protein and secretory IgA. Four weeks after immunization, the vaccinees and a matched group of control subjects were inoculated with attenuated A/England/42/72 (MRC-7) virus. Evidence of infection was found in 14 of 23 (61 per cent) of control subjects and in seven of 23 (30 per cent) of immunized volunteers. This result showed a significant protection (P = 0.04) against challenge virus infection for volunteers given intranasal vaccine.     

Attempts at detection of actomyosin associated with influenza virus

Influenza virus strans A/Scotland/74, A/Hong Kong/68, A/Port Chalmers/73 and the MRC-12 recombinant were tested with immune antiserum against actomyosin. As shown by electron microscopy, the serum aggregated virus particles, but only after bromelain treatment (without haemagglutinin and neuraminidase spikes). In rocket electrophoresis the serum gave positive precipitation reaction with all the strains tested, and with virus from various hosts (chick embryo, monkey kidney cell culture, mice after adaptation). There fore the host protein presumably is present in the influenza virus structure irrespective of the strain or the host in which the virus is grown.     

A contribution of cellular immunity to protection against influenza in man

The degree of lymphocyte transformations and leukocyte migration inhibition (LMI) in the presence of inactivated A/Scotland/74 (H3N2) influenza virus vaccine was measured in blood samples collected from 56 medical student volunteers. At the same time the volunteers were skin tested, using the same vaccine. Using the antigenically similar WRL 105 (H3N2), recombinant influenza virus, the level of haemagglutination-inhibiting (HI) antibodies in serum, and neutralizing antibodies in nasal washings collected from the volunteers, were also determined. Each volunteer was then inoculated with live, attenuated WRL 105 influenza virus vaccine and infections demonstrated by virus isolations and serology.Correlations between the ability to infect the volunteers and the various parameters of humoral and cellular immunity were then determined. The results showed a good correlation between the level of serum HI antibody and infection. Thus 16 of 20 volunteers with serum HI antibody titres of 110, but only 6 of 20 volunteers with antibody levels of 130, showed evidence of infection. No direct correlation was observed between any of the other parameters measured and infection by WRL 105 virus. However, when the LMI and serum HI antibody levels were considered together, a contribution of cellular immunity, as measured by the LMI test, could be found. Of 19 volunteers with low serum HI antibody and low LMI levels, 16 were infected, whereas of 13 volunteers with low HI antibody, but with high LMI levels, only 6 showed evidence of infection with WRL 105 influenza virus.     

Attenuated influenza produced by experimental intranasal inoculation.

To assess the relative effect of natural versus experimental influenza illness on pulmonary function, we compared 43 normal adults with documented nonpneumonic influenza A infection during three outbreaks, 1974 (A/Port Chalmers/74), 1975 (A/Port Chalmers/74), and 1976 (A/Victoria/75) to 24 normal volunteers following nasal inoculation with wild-type influenza A/England/42/72, A/Scotland/74 or A/Victoria/75. In naturally acquired illness, abnormalities in small airway functiion and transiently increase airway reactivity were observed. In contrast, no such dysfunction was observed in experimentally induced illness. This group manifested milder illness and significantly shorter duration of cough.     

Lymphocyte cytotoxicity to influenza virus-infected cells: response to vaccination and virus infection

Peripheral blood leukocytes obtained from volunteers at various times following influenza vaccine or live influenza virus infection were assayed for cytotoxicity against influenza virus-infected cells. Cytotoxicity was highest on days 3 and 7 following vaccination with commercial A/Port Chalmers/1/73 inactivated influenza virus vaccine. Maximal cytotoxicity was found 9 days after infection induced by intranasal inoculation of a strain of A/Scotland/840/74 influenza virus. Individuals naturally infected with A/Victoria/3/75 were also found to have elevated cytotoxicity approximately 1 week after onset of illness. Cytotoxicity levels decreased toward base line approximately 30 days after the virus exposure. The effector mechanism(s) responsible for the early, transient elevation in specific immune release to influenza virus-infected cells may be different from the antibody-dependent cell cytotoxicity demonstrated in the peripheral blood leukocytes from volunteers who had a remote experience with influenza virus.     

A clinical trial with Alice/R-75 strain,live attenuated serum inhibitor-resistant intranasal bivalent influenza A/B vaccine

A clinical trial was conducted with Alice/R-75 strain live attenuated intranasal influenza A/B vaccine. With double blind control 88 adult volunteers were administered 2 doses of Alice/R-75 vaccine, 93 volunteers received one dose of Alice/R-75 vaccine and one dose placebo solution and 94 subjects were administered 2 doses of placebo solution. Twenty-three other subjects received Alice strain monovalent influenza A vaccine. For comparison, data from 21 subjects who received monovalent intranasal R-75 strain influenza B in two doses is included. The vaccine was generally well tolerated. Four-fold serum hemagglutination-inhibiting (HAI) antibody titer rises to A/England/42/72 occurred in 39% of the monovalent Alice strain vaccinees; in contrast 18% of those given 2 doses of bivalent Alice/R-75 vaccine and 11% of those given 1 dose of bivalent vaccine had similar four-fold HAI antibody titer rises. HAI antibody titer rises to influenza B/Hong Kong/72 occurred in 38% of R-75 strain monovalent vaccinees, 14% of Alice/R-75 2-dose vaccinees and 11% of Alice/R-75 one dose vaccinees. An epidemic of influenza at the onset of the study made evaluation of the efficacy of the vaccine impossible.This study was supported by a grant from Smith, Kline and French Laboratories, Philadelphia, Pennsylvania     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Regional T- and B-cell responses in influenza-infected ferrets.

Ferrets were infected with A/Port Chalmers/72 influenza virus and the T- and B-cell responses in the spleen, in lymph nodes draining the upper and lower respiratory tract, and in lung washings were examined in vitro. Lymphocyte responses were measured by using a hemolytic plaque assay for B cells and a proliferation assay for T cells. Virus and antibody levels were measured in respiratory tract washings, and antibody titers were measured in sera from infected animals. Individual B cells secreting specific antibody to A/Port Chalmers/72 virus were detected in regional lymph node and spleen preparations as early as 3 days and as late as 43 days after infection. T-cell assays showed an in vitro response of lymph node cells to A/Port Chalmers/73 virus from day 6 to day 43. Virus was isolated from the respiratory tract up to 7 days after infection. Serum hemagglutination-inhibiting antibody was first detectable on day 6, with maximum titers reached by day 10. These results demonstrated that antibody production and a cellular immune responses were detectable at regional sites at a time when virus was still present and before serum antibody was measured.     

Use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza A virus vaccines.

Sera from volunteers who received live influenza A wild-type or ts recombinant virus were tested by hemagglutination inhibition (HI) assay, neuraminidase inhibition (NI) assay, and the enzyme-linked immunosorbent assay (ELISA) to determine which assay system was the most sensitive in detecting an immunological response to infection. The ELISA was performed with inactivated whole virus antigen, and the optical density at each of five serial twofold dilutions of pre- and postimmunization sera was measured. The difference in the amount of ELISA antibody in pre- and postinoculation serum specimens was taken to be proportional to the area between the respective titration curves. The ELISA was more sensitive than the HI or NI test in detecting a seroresponse in volunteers infected with A/Hong Kong/123/77 (H1N1), A/New Jersey/8/76 (Hswine N1), or A/Alaska/6/77 (H3N2) ts recombinant virus. These results suggest that the ELISA should be used to determine the frequency of infection with attenuated viruses as well as the 50% human infectious dose of candidate live influenza A vaccine viruses.