Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice

Subunit and intact influenza A virus vaccines have been compared with infectious virus in a mouse model for their ability to induce memory for cross-reactive cytotoxic T cell responses and to protect mice from challenge with different subtypes of influenza A virus. There is an overall correlation between secondary cytotoxic T cell responses and cross-protection. The most long-lasting and successful cross-protection was observed after intranasal infection with influenza virus A/X31 (H3N2) that replicates efficiently in mice and induces high levels of memory for cross-reactive cytotoxic T cell responses. Short-lasting cross-protection and low levels of T cell-mediated cytotoxicity were associated with infection by A/USSR (H 1N1) virus, that replicates to lower titers in mice, or after multiple injections of inactivated whole virus vaccine. No cross-protection to challenge with heterologous influenza virus was detectable after 1-2 injections of HANA influenza subunit vaccine which failed to prime hosts for cytotoxic T cell responses. These findings may have important implications for vaccination strategy. If cytotoxic T cells play a role in the protection of humans from influenza, live attenuated vaccines should be considered instead of the currently recommended inactivated virus or subunit vaccines.     

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA-, A/Yamagata HA-, and A/Fukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount detected in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A/Fukuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.     

Clearance of antibody and complement in serum after challenge of mice previously vaccinated or infected with influenza virus.

Mice previously vaccinated or convalescent after infection with mouse-adapted influenza virus were challenged by various routes with live or inactivated virus. The levels of haemagglutination inhibiting and complement-fixing antibodies and complement in serum were found to decrease distinctly within several hours after challenge: the reconstitution to initial levels occurred within three days.     

Selective suppression of the cytotoxic T cell response to influenza virus in mice

Mice injected with inactivated (UV light-irradiated) influenza virus produce specific antibody, become sensitized for a delayed-type hypersensitivity reaction, but do not generate specific cytotoxic T (T_c) cells. If injected 4–5 days later with infectious virus, the formation of T_c cells is suppressed by > 90%. If A strain viruses are used, the suppression observed is cross-reactive within A strain viruses but does not extend to B/LEE or to Sendai virus. Serum from mice injected with UV-irradiated virus contains antibodies which on adoptive transfer can inhibit T_c cell formation when infectious homologous virus is used to challenge the recipients. Spleen cells from the same mice, upon adoptive transfer, also inhibit (50–70%) T_c cell formation if transferred within 24 h of injection of infectious virus, and the specificity pattern observed is cross-reactive within A strains. The activitiy of the cells mediating suppression is destroyed by monospecific anti-Thy-1.2 antibody and complement. The immune cells require I region sharing between donor and recipient mice for their suppressor activity to be effective. (There is also a partial requirement for K. D region sharing, but the possible rejection of transferred cells is not excluded.) Dilution assays in which clonal expansion of T_c precursors is used to estimate their frequency and the presence of T helper (T_h) cells indicate that suppressed mice possess T_c precursors and primed cells which, upon restimulation, act as T_h cells. Furthermore, injection of irradiated T_h cells with inactivated virus does not significantly reduce the ensuing suppression.     

Mast cell degranulation is induced by A549 airway epithelial cell infected with respiratory syncytial virus

Respiratory syncytial virus (RSV), a major causative agent of respiratory tract infections, influences allergic diseases. Mast cells, important effector cells in allergic disease, also express chemokine (C-X₃-C motif) receptor 1 (CX₃CR1). The RSV attachment glycoprotein (G protein) is structurally similar to CX₃C ligand 1 (CX₃CL1), the CX₃CR1 ligand, suggesting that RSV directly interacts with and affects mast cell function, including degranulation. In this paper, the effect of RSV infection on mast cell function was studied using the human mast cell line (HMC-1). The results showed that RSV infection and replication was inefficient in HMC-1 cells than in human epithelial A549 cells. Additionally, HMC-1 degranulation occurred only in coculture with RSV-infected A549 cells, with up-regulation of TNFα secretion. However, direct RSV inoculation and incubation with RSV-infected A549 cell culture medium failed to induce HMC-1 degranulation, suggesting that virus-infected cells are critical for degranulation during RSV infection; however, degranulation does not occur by direct RSV infection into mast cells.     

Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus

In mice acutely infected with respiratory syncytial virus (RSV), more than 20% of pulmonary CD8(+) T cells, but only 2-3% of CD8(+) T cells in the draining lymph node secreted interferon-gamma in response to a single peptide. Surprisingly, the percentage of virus-specific T cells in the lung remained at these high levels long after the acute infection. Pulmonary memory T cells were further studied in a sensitive adoptive transfer system, which allows visualizing polyclonal CD4(+) and CD8(+) virus-specific memory T cell responses. Fifty days after infection, persisting RSV-specific pulmonary T cells remained CD69(hi) CD62L(lo), but had returned to a resting memory state according to functional criteria. In the absence of neutralizing antibodies reinfection first induced cell division among virus-specific memory T cells 3 days after infection predominantly in the local lymph node. However, divided cells then rapidly accumulated in the lung without significantly increasing in the lymph node. These results suggest rapid export of reactivated cells from the lymph node to the target organ. Thus, although memory T cells can be maintained in the infected organ after a localized virus infection, amplification of a recall response appears to be most effective in organized lymphoid tissue.     

LIGHT is dispensable for CD4+ and CD8+ T cell and antibody responses to influenza A virus in mice

The tumor necrosis factor family ligands, LIGHT (lymphotoxin like, exhibits inducible expression and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), 4-1BBL and CD70, are found in the same gene cluster on mouse chromosome 17. Although the roles of 4-1BB-4-1BBL and CD27-CD70 interactions in anti-viral T cell responses have been well established, the role of LIGHT in T cell activation/expansion in vivo is less clear. Under conditions that were previously employed to demonstrate a role for 4-1BBL in CD8+ T cell memory, wild-type and LIGHT-/- mice were infected with influenza A virus and primary and memory/recall responses were measured at various time points thereafter. Neither primary expansion nor memory/recall CD8+ T cell responses were affected by the absence of LIGHT, as measured up to 2 months post-infection. CD4+ T cell responses were also unaffected by LIGHT deficiency. Furthermore, we found that LIGHT played no role in the induction of influenza-specific IgG1 and IgG2a serum antibodies. Taken together, these data suggest that LIGHT is dispensable for the acquired immune response to influenza virus in mice with no effect on the induction, maintenance or reactivation of CD8+ T cell memory.     

Enhanced protection against respiratory influenza A infection in mice by liposome-encapsulated antibody.

Liposome-mediated passive immunity was evaluated for its efficacy in the prophylaxis and treatment of influenza A/PR/8 virus infection in mice. A mouse LD50 protection model was developed using a polyclonal anti-influenza A antibody which demonstrated strong reactivity against the mouse-adapted virus in a fluorogenic enzyme immunoassay and in an in vitro plaque assay. Using liposomes as an antibody carrier system, the delivery of antibody to the lungs was optimized. For mice given the antiviral antibody intranasally 24 h prior to challenge with 10 LD50 of mouse-adapted influenza A/PR/8 virus, the survival rate at 14 days post-challenge was 60%. However, when mice were given antibody encapsulated within liposomes, the survival rate increased to 100%. In the treatment of mice preinfected with 10 LD50 of the virus, mice were fully protected (100% survival rate) when treated within 8 hr post-infection with free unencapsulated antibody, or within 12 hr with liposome-encapsulated antibody. It is postulated that the improved therapeutic and prophylactic efficacies of the antiviral antibody may be attributed to enhanced delivery as well as retention of antibody molecules in the lungs when liposomes are used as antibody carrier system.     

Differences in antibody responses of mice to intranasal or intraperitoneal immunization with influenza A virus and vaccination with subunit influenza vaccine

Two antigenically related but different influenza A virus strains of H3N2 subtype, A/Dunedin/ 4/73 (H3N2) (Dunedin) and A/Mississippi/1/85 (H3N2) (Mississippi), were used for intranasal (i.n.) and intraperitoneal (i.p.) immunization of mice and respective antibody responses were compared. In ELISA, using purified influenza A virus as antigen, the highest titer of antiviral antibodies was observed after a repeated i.n. infection, in which the Dunedin strain was followed by the Mississippi strain and vice versa. Similarly, in virus neutralization (VN) test, the highest titer of VN antibodies was found after a repeated i.n. infection. The subunit vaccine INFLUVAC, when administered intramuscularly (i.m.), induced only a poor antibody response as assayed by ELISA. Moreover, the INFLUVAC vaccination elicited a 100-fold lower titer of VN antibodies than the i.n. infection and an approx a 10-fold lower titer than the i.p. immunization. A repeated INFLUVAC vaccination did not lead to a significant increase of VN antibody titer. Also the antibody response to HA2gp--a conserved part of influenza hemagglutinin (HA) that might contribute to the induction of specific antiviral antibodies--was followed. Similarly to the VN antibody response, the highest HA2 antibody titer was induced after a repeated i.n. infection, whereas the lowest HA2 antibody titer was observed after a single or repeated INFLUVAC vaccination. Overall, the HA2 antibody titers remarkably well corresponded to the VN potential of the examined sera.     

Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes

CD8+ cytotoxic T lymphocytes (CTLs) contribute to the control of viral infections by recognizing peptides of viral proteins presented by MHC class I molecules on infected cells. Some viruses have developed strategies to evade recognition by CTL. One of these strategies involves antigenic variation in CTL epitopes as described for viruses chronically infecting their host like EBV, HIV, HBV and HCV. Here we show three examples of variation in CTL epitopes in the influenza virus nucleoprotein (NP) associated with escape from CTL immunity. The first two involve a mutation at position 384 of the NP, which is the anchor residue of a HLA-B*2705-restricted epitope NP383-391 (SRYWAIRTR) and the HLA-B*08-restricted epitope NP380-388 (ELRSRYWAI). It was shown that these mutations have arisen in the 1993/1994 season and that these mutant variants completely replaced the virus strains containing the wild-type epitopes. Furthermore, T cell recognition was completely abrogated by the R384G mutation. A third example of variation in an influenza virus CTL epitope was found in a newly identified HLA-B*3501-restricted CTL epitope. This immunodominant epitope exhibited extensive amino acid sequence variation and the variants emerged in a chronological order. Again CTL specific for older variants failed to recognize more recent strains of influenza A virus, indicating an escape from CTL immunity. Thus, in addition to the introduction of mutations in the surface glycoproteins like the hemagglutinin, allowing escape from antibody-mediated immunity, there is now evidence that influenza viruses can escape in a similar way from CTL-mediated immunity.     

Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus

Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma), play an important role in defending against influenza infection. However, there is an age-associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mphi), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E-supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0.02 and 0.001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)-2 and IFN-gamma production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL-2 and IFN-gamma in old mice; higher IFN-gamma production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)-stimulated Mphi PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL-6, IL-1beta, or tumour necrosis factor-alpha (TNF-alpha) production by splenocytes. Young mice fed the vitamin E-supplemented diet had significantly lower IL-1beta (day 7) and TNF-alpha production (day 5) compared with those fed the control diet. Old mice fed the vitamin E-supplemented diet had significantly lower TNF-alpha production (day 2) than those fed the control diet. Our results indicate that the vitamin E-induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.     

Changes in the protease activity in the lungs of mice infected with the influenza A virus

In uninfected animals, the level of protease and protease-inhibiting activities in the serum are in balance which is broken after influenza A virus infection. Most profound changes occur within the first few hours after infection. In 6 hours postinfection the amount of protease decreases both in the lungs and serum of the infected animals, and the protease-inhibiting activity increases. In the period of the highest accumulation of the infectious virus 2 days after infection, proteolytic activity also decreases, this decrease coinciding with that of the inhibiting activity. The third period of increased protease activity also coincides with amplification of infectious virus progeny and appears to be associated with consequences of virus infection and bacterial superinfection.     

The sensitization of mice with a wild-type and cold-adapted variant of influenza A virus. II. Secondary cytotoxic T cell responses.

Reductions in virus titres and the generation of enhanced cytotoxic T cell (Tc) activity in the lungs of mice primed either with a wild-type, parental (H2N2) influenza virus, A/AA/6/60, or a cold-adapted variant A/AA/6/60-ca and challenged 6 weeks later with a H1N1 A/WSN virus showed that both H2N2 viruses could sensitize the mice. A comparison of graded sensitizing doses of each virus showed that inocula of 10(6) tissue culture infective doses (TCID50) of the ca-variant or 10(3) TCID50 of the wild-type virus gave similar results. The spleens and lungs of normal mice were found to contain similar levels (circa 1/10(5) cells) of precursor Tc cells and the level in the lung did not increase 2 days after intranasal (i.n.) inoculation of A/WSN virus. Two and 6 weeks after priming mice with 10(5) TCID50 of either virus, the lungs contained about a 20-fold increase in the precursor Tc cell frequency. In contrast, sensitization with a sub-lethal dose of a mouse-adapted A/WSN virus caused a 100-fold or greater increase. Sensitization of mice with the parental but not the ca-variant virus caused an increase in frequency of precursor Tc cells in the spleens of the sensitized mice and this might reflect the very low level of replication of the ca-variant virus in the mouse lung.     

Systemic and local antibody responses in elderly subjects given live or inactivated influenza A virus vaccines.

Intranasal live attenuated cold-adapted (ca) influenza A/Kawasaki/9/86 (H1N1) reassortant virus and parenteral inactivated influenza A/Taiwan/1/86 (H1N1) virus were given alone or in combination to 80 ambulatory elderly subjects. An enzyme-linked immunosorbent assay was used to measure hemagglutinin-specific (HA) antibodies in serum and nasal wash specimens collected before vaccination and 1 and 3 months later. Serum immunoglobulin G (IgG) and nasal wash IgA HA responses were elicited in 56 and 20%, respectively, of 25 inactivated-virus vaccinees and in 67 and 48%, respectively, of 27 recipients of both vaccines but in only 36 and 25%, respectively, of 28 vaccinees given live virus alone. Inactivated virus, administered alone or with live virus vaccine, induced higher titers of serum antibody than did the live virus alone. In contrast, nasal IgA HA antibody was elicited more often and in greater quantity by the vaccine combination than by either vaccine alone. Despite these differences, the peak titers of local antibody mounted by each group of vaccinees were similar. By 3 months postvaccination, serum IgG and nasal IgA HA antibody titers remained elevated above prevaccination levels in 50 and 17%, respectively, of the inactivated-virus vaccinees and in 46 and 23%, respectively, of recipients of both vaccines but in only 19 and 7%, respectively, of the live-virus and systemic antibodies, if vaccinees. The finding that live ca influenza A virus induced short-lived local and systemic antibodies, if confirmed, suggests that live virus vaccination may not be a suitable alternative or adjunct to inactivated virus vaccination for the elderly.     

Diminished T cell surveillance function in old mice infected with lymphocyte choriomeningitis virus.

The cell-mediated immune response resulting from infection with lymphocytic choriomeningitis virus (LCMV) was compared in old (18--20 month) and young (4--6 month) random-bred ICR mice. Three separate assay systems were used: the level of virus-specific cytotoxic T-cell activity measured in vitro, time of onset of fatal cell-mediated immunopathology in vivo and adoptive transfer of inflammatory process to immunosuppressed, virus-infected recipients. The capacity of old mice to generate virus-immune effector T cells was, by all of these criteria, greatly diminished. It is suggested that the LCMV-specific T-cell response may prove a useful standard system for determining integrity of immunological surveillance function.     

Reconstitution of the cellular immune response by lactoferrin in cyclophosphamide-treated mice is correlated with renewal of T cell compartment

Cyclophosphamide is an alkylating agent used to treat both malignant and non-malignant immune-mediated inflammatory disorders in humans. It is also known as a potent immunosuppressive drug in humans and experimental animals. The aim of this study was to evaluate the effects of oral administration of lactoferrin (LF) on cellular responses and reconstruction of the lymphocyte pool in mice treated with cyclophosphamide (CP). Twelve week-old CBA mice were given a single intraperitoneal (i.p.) dose of CP (400 mg/kg body weight), then were treated per os with seven doses of LF (1 mg/dose) on alternate days. We demonstrated that the magnitude of delayed type hypersensitivity to ovalbumin, strongly diminished by CP action, was reconstituted by LF. Oral LF treatment also resulted in partial recovery of Concanavalin A-induced splenocyte proliferation. Blood profile analysis revealed elevation of leukocytosis by LF in CP-treated mice (from 64.9 to 84.76% of the control value). LF also caused substantial restoration of the percentage of the lymphocyte population in circulating blood (from 43.4 to 60.2% of the control values). LF alone had no effect on the neutrophil/lymphocyte ratio in normal mice, however, the total number of leukocytes decreased by 23.25%. Furthermore, we showed that LF increased the cellularity of spleens isolated from CP-treated mice (from 53.2 to 78.8%) and the content of peritoneal and alveolar macrophages (elevations from 50.6 to 67.3% and from 65.2 to 83.6%, respectively). Lastly, using panning technique, we demonstrated that LF strongly elevated the pool of CD3+ T cells in normal and CP-immunocompromised mice and CD4+ T cell content. In conclusion, we showed for the first time that lactoferrin, given orally to CP-immunosuppressed mice, could reconstitute a T-cell mediated immune response by renewal of the T cell pool.