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.     

The effect of bacterial peptidoglycans on the immune response of hamsters to influenza virus vaccines

The immune response of hamsters to influenza virus vaccine incorporating peptidoglycans derived from Streptomyces griseus was investigated. The results showed that although inoculation of a subunit influenza virus vaccine together with a peptidoglycan elicited a markedly increased serum HI antibody response compared to that induced by the subunit vaccine alone, pre-treatment of animals with peptidoglycans resulted in a suppression of the antibody response to the vaccine haemagglutinin antigen. The immunosuppressive effect was dose-related, and could be shown by adoptive transfer experiments to be transferred by spleen cell preparations from peptidoglycan-treated hamsters. The reasons for these findings, and their implication for the use of peptidoglycans as 'carriers' in influenza virus subunit vaccines, are discussed.     

Reactogenicity and immunogenicity of a surface-antigen-adsorbed influenza virus vaccine in children.

An influenza virus vaccine containing the purified surface haemagglutinin and neuraminidase antigens of A/Victoria/75 and B/Hong Kong/73 viruses adsorbed to an aluminium hydroxide gel was assessed for reactogenicity and immunogenicity in children aged 4 to 11 years, since there is no influenza virus vaccine available for this age group. Significant serum haemagglutination-inhibiting antibody responses to the A/Victoria/75 and B/Hong Kong/73 haemagglutinin antigens present in the vaccine were observed in 47% and 35%, respectively, of the children vaccinated, with a single dose. The vaccine induced no significant local or systemic reactions.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

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.     

Antibody reactive in antibody-dependent cell-mediated cytotoxicity following influenza virus vaccination

The antibody reactive in antibody-dependent, cell-mediated cytotoxicity (ADCC) to influenza virus-infected cells was measured in two groups of seven volunteers each, before and after immunization with inactivated or live attenuated A/Victoria/3/75 influenza virus vaccines. Age-matched controls were seven adult individuals who experienced natural influenza infection due to A/Victoria/3/75-like virus strain. After inactivated whole influenza virus immunization all the subjects showed a significant rise of the antibody reactive in ADCC (from a mean value of 4.7% to 17.1% cytotoxicity, before and 5 weeks after immunization, respectively) as well as of hemagglutination inhibition (HI) antibody (fourfold or greater increase). These immune responses were similar to those observed among naturally infected controls. After live attenuated virus vaccination, no significant increase in titer of antibody reactive in ADCC was detected, even though the vaccine induced significant increase of HI antibody titer. Little correlation was found between ADCC and HI antibody rises in sera of recipients of inactivated virus vaccine and of naturally infected individuals, while, in live attenuated influenza virus vaccinees, the rise of HI antibody titer did not correspond to a significant increase of ADCC antibody titer; several subjects who developed a significant rise in ADCC antibody titer did not show significant variation in antibody to neuraminidase and/or to complement fixation influenza virus antigens.     

Safety of and serum antibody response to cold-recombinant influenza A and inactivated trivalent influenza virus vaccines in older adults with chronic diseases.

Forty older adults with chronic diseases were vaccinated intranasally with either influenza A/California/10/78 (H1N1) (CR37) or influenza A/Washington/897/80 (H3N2) (CR48) virus. No clinically significant morbidity or decrement in pulmonary function occurred postvaccination. Two (15%) recipients of CR37 virus and twelve (44%) recipients of CR48 virus became infected with vaccine virus, as indicated by a fourfold rise in serum hemagglutination inhibition antibody titer; a fourfold rise in serum immunoglobulin G (IgG) or IgA antibody titer, indicated by enzyme-linked immunosorbent assay; isolation of vaccine virus from nasal washings; or all of these. Within 1 year after cold-recombinant vaccine virus vaccination, 18 vaccines received inactivated trivalent influenza virus vaccine parenterally. Of the vaccinees, 13 (72%) developed a fourfold rise in serum antibody titer to H1N1 antigen and 16 (89%) developed a fourfold rise in serum antibody titer to H3N2 antigen. We conclude that administration of these cold-recombinant vaccine viruses to older adults with chronic diseases was safe, but that serum antibody response rates were lower than those achieved with subsequently administered inactivated influenza virus vaccine given parenterally. However, the higher seroconversion rates attained by using the inactivated trivalent influenza virus vaccine do not necessarily mean that it is more efficacious in preventing infection or severe illness or both due to natural wild-type influenza A virus.     

The immune response to influenza vaccines

Specific immunity to influenza is associated with a systemic immune response (serum haemagglutination inhibition antibody), local respiratory immune response (virus-specific local IgA and IgG antibodies in nasal wash), and with the cell-mediated immune response. Both inactivated and live influenza vaccines induce virus-specific serum antibody which can protect against infection with influenza virus possessing the same antigenic specificity. In the absence of serum antibodies, local antibodies in nasal wash are a major determinant of resistance to infection with influenza virus. In comparative studies in humans it was shown that nasal secretory IgA develops chiefly after immunization with live cold-adapted (CA) vaccine, but persistent nasal secretory IgG was detected in both CA live and inactivated vaccines. The origin of nasal wash haemagglutination inhibition (HI) antibodies is not completely known. Recently it was found that cytotoxic T-cells (CTL) play an important role in immunity against influenza and in clearance of influenza virus from the body. In primed humans, inactivated influenza vaccine stimulates a cross-reactive T-cell response, whereas the ability of inactivated vaccine to stimulate such immunity in unprimed humans has not been determined. Data on the T-cell response to live vaccine in humans are limited to the development of secondary T-cell responses in primed individuals vaccinated with a host-range (HR) attenuated vaccine. The data obtained have shown that immunity induced by inactivated influenza vaccines is presumably dependent on the stimulation of serum antibody. Live CA vaccines not only stimulate a durable serum antibody response, but also induce long-lasting local respiratory tract IgA antibody that plays an important role in host protection.     

Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)-specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.     

E-rosette forming cells and humoral antibody titres in humans after vaccination with three different inactivated influenza virus vaccines A/USSR/92/77 (H1N1)

The number of E-rosette forming cells and the serum haemagglutination inhibition (HI) antibody titres were examined in 37 volunteers immediately before and 14, 28, 35 and 63 days after immunization with three inactivated influenza virus vaccines A/USSR/92/77 (H1N1)--NIB 6 and in 11 non-vaccinated controls. From the former, 10 volunteers were immunized with 1000 haemagglutinin (HA) IU per dose, 11 volunteers with the NIB 6 adsorbate vaccine (340 HA IU/dose) and 16 volunteers with a bivalent vaccine composed of 180 HA IU/dose NIB 6 and 180 HA IU/dose of influenza virus A/Bangkok X-73 (H3N2). The percentage of E-rosette forming cells was decreased in all vaccinated volunteers 14 days after vaccination; later on the values reached normal level of non-vaccinated controls or of subjects before vaccination. The number of E-rosette forming cells was in correlation with the applied virus vaccine dose, i.e. for the 1000 HA IU/dose: 29.95 +/- 11.74%, p less than 0.001 and for the 340 HA IU/dose: 47.75 +/- 11.15%, p less than 0.005; however, after administration of 180 HA IU/dose of NIB 6 in the bivalent vaccine, the value 58.65 +/- 11.5% was not significantly decreased in comparison to non-vaccinated donors. The serum HI antibody titres reached the highest level 14 days after vaccination and remained constant during the next 6 weeks. There was a correlation between decreased E-rosette values and increased serum antibody titres (p less than 0.05). The current study indicates that the number of E-rosette forming cells may serve as a further laboratory criterion for controlling the effect of inactivated influenza virus vaccines on the immune system of man.     

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.     

Radioimmunoassay of influenza A virus haemagglutinin. II. Antigenic cross-reactions of influenza A (H3 subtype) viruses as determined by radioimmunoassay and haemagglutination inhibition tests

Individual rabbits differed greatly in their antibody response to the "strain-specific" and "cross-reactive" antigenic determinants on the haemagglutinin (HA) subunit of influenza virus recombinant MRC11 (H3N2) and influenza virus Dunedin (H3N2), after immunization with whole virus or bromelain-released haemagglutinin (B-HA). Consequently, diverse cross-reactions between htese viruses and A/Hong Kong/68 virus were found in the haemagglutination inhibition (HI) test as well as in homologous radioimmunoassay (125I-B-HA from MRC11:anti MRC11 serum, and 125I-B-HA from Dunedin: anti Dunedin serum) when sera from different animals were employed. Radioimmunoassay (RIA), over and above to the HI test, was able to differentiate clearly the respective HAs also with antisera reacting to the same HI titre with both corresponding influenza virus strains. Thus it appeared that antigenic differences could be identified with higher sensitivity by homologous RIA than by the HI test and that multiple antigenic determinants were reactive on the 125I-B-HA in the RIA procedure employed. MRC11 and A/HK/68 viruses were also compared by heterologous RIA (125I-B-HA from MRC11: anti A/HK/68 serum). It was found that preferentially antigenic determinants with a high degree of cross-reactivity could be studied in the heterologous system.     

Use of the enzyme-linked immunosorbent assay (ELISA) for the estimation of serum antibodies in an influenza virus vaccine study

The value of the enzyme-linked immunosorbent assay (ELISA) for de termining the serum antibody responses of volunteers following immunisation with various inactivated influenza virus vaccines was assessed, and the incidence of seroconversions, as measured by both haemagglutination-inhibition (HI) and ELISA response of the volunteers determined. ELISA was found to be more sensitive than the HI test in detecting serum antibodies, but was also less specific under the conditions used. With regard to efficacy, the whole virus vaccine proved to be more effective in inducing serum antibody in an unprimed population than either tween-ether split or subunit adsorbed vaccines, but the reverse situation held when the population was primed with respect to the antigen concerned.     

A comparative study of the inoculation properties of live recombinant and inactivated influenza vaccines made from strain A/Philippines/2/82 (H3N2) in 8- to 15-year-old children]

This study was carried out to compare reactogenicity, immunogenicity, and efficacy of live attenuated and inactivated influenza vaccines prepared from influenza A/Philippines/2/82-like virus strains. Schoolchildren of a boarding school of Moscow were randomly divided into three groups: (1) vaccinated with a live attenuated vaccine, (2) vaccinated with inactivated influenza vaccine, and (3) given placebo. Both vaccines were well tolerated by the children, with practically no severe general or local reactions. The inactivated vaccine was found to be superior to the live one in its capacity to stimulate humoral immunity studied by HI, EIA, and microneutralization tests. In 69.7% of the children given the inactivated vaccine, seroconversion to the vaccine strain was detected by two or three methods of antibody titration used. Only 35.4% seroconversions were demonstrated in children immunized with the live influenza vaccine. Enzyme immunoassay was found to be a more sensitive but less specific method for antibody titration as compared with HI test whereas microneutralization proved to be more specific but less sensitive for titration of antibodies to influenza A (H3N2) viruses.     

CD40-mediated enhancement of immune responses against three forms of influenza vaccine

There is potential for influenza A infections to cause massive morbidity and mortality. Vaccination may be the primary defence against pandemic influenza, and potential pandemic'flu vaccines may be produced conventionally, in embryonated eggs, or as recombinant protein or synthetic peptide vaccines. However the vaccines are produced, the supply may be limiting, and it will be important to enhance the immunogenicity of the vaccines as much as possible. We have shown that conjugation to CD40 binding antibody is a very efficient way of enhancing immune responses against model antigens, but were interested in assessing the effectiveness of this system using influenza vaccines. We produced conjugates of CD40 monoclonal antibody (mAb) and isotype control with three potential influenza vaccines: a peptide-based vaccine containing T- and B-cell epitopes from virus haemagglutinin; a whole, killed virus vaccine; and a commercially produced split virus vaccine. CD40 mAb conjugates in each case were more immunogenic, but the adjuvant effect of CD40 conjugation was greatest with the split vaccine, where antibody responses were enhanced by several hundred-fold after a single immunization, and lymphocyte proliferation in response to antigen in vitro was also strongly enhanced.     

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.     

Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants

Recombinant vaccinia viruses enable studies of immune recognition of antigens expressed from single viral genes. We have constructed recombinants expressing the haemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus A/PR/8/34 (H1N1). These recombinant viruses together with a recombinant expressing the HA from influenza virus A/JAP/305/57 (H2N2) have been used to examine the cytotoxic T lymphocyte (CTL) response to these influenza virus antigens. Both antigens are recognised by murine CTL and recognition of HA is influenza virus subtype-specific, whereas recognition of NP is crossreactive. In limiting dilution studies approximately 10% of the influenza CTL response is HA-specific, while approximately 30% of the response is NP-specific. Despite the ability of NP to stimulate a significant CTL response, mice immunised with the NP-vaccinia recombinant are not as well protected from subsequent lethal challenge with influenza virus, as mice immunised with the HA vaccinia recombinant. These studies demonstrate that viral antigens expressed from vaccine recombinants can provide protective immunity and that the influenza-poxvirus recombinants can provide data on protective immunity generated by individual viral proteins.     

Comprehensive serological analysis of two successive heterologous vaccines against H5N1 avian influenza virus in exotic birds in zoos

In 2005, European Commission directive 2005/744/EC allowed controlled vaccination against avian influenza (AI) virus of valuable avian species housed in zoos. In 2006, 15 Spanish zoos and wildlife centers began a vaccination program with a commercial inactivated H5N9 vaccine. Between November 2007 and May 2008, birds from 10 of these centers were vaccinated again with a commercial inactivated H5N3 vaccine. During these campaigns, pre- and postvaccination samples from different bird orders were taken to study the response against AI virus H5 vaccines. Sera prior to vaccinations with both vaccines were examined for the presence of total antibodies against influenza A nucleoprotein (NP) by a commercial competitive enzyme-linked immunosorbent assay (cELISA). Humoral responses to vaccination were evaluated using a hemagglutination inhibition (HI) assay. In some taxonomic orders, both vaccines elicited comparatively high titers of HI antibodies against H5. Interestingly, some orders, such as Psittaciformes, which did not develop HI antibodies to either vaccine formulation when used alone, triggered notable HI antibody production, albeit in low HI titers, when primed with H5N9 and during subsequent boosting with the H5N3 vaccine. Vaccination with successive heterologous vaccines may represent the best alternative to widely protect valuable and/or endangered bird species against highly pathogenic AI virus infection.     

Enhanced response to influenza a vaccines in hamsters primed by prior heterotype influenza infection

Summary The serum haemagglutination-inhibiting antibody response of hamsters to immunization with inactivated monovalent influenza vaccines is enhanced if the animals are subjected to a prior infection with a live, heterotypic influenza virus.     

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.