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.     

Viral neuraminidase treatment of dendritic cells enhances antigen-specific CD8(+) T cell proliferation, but does not account for the CD4(+) T cell independence of the CD8(+) T cell response during influenza virus infection

In vitro studies demonstrate that the increased alloreactive T cell response to dendritic cells (DC) that are treated with either live or inactivated influenza virus A/PR/8/34 is due to viral neuraminidase (NA) activity. Since virus-specific cytotoxic T lymphocytes (CTL) play an important role in immunity to heterologous influenza strains, we compared the activation of CD8(+) T cells by untreated and NA-treated DC. Increased CTL activity was induced by NA-treated DC both in vitro and in vivo. Since the generation of CTL in response to influenza virus infection does not require prior "activation" of DC by CD4(+) T cells (as is the case for many antigens), we asked whether NA activity contributed to this unconditional CD8(+) T cell response. This was not the case. Future studies will determine the factors that are responsible for the CD4(+) T-cell-independent influenza virus-specific CTL response.     

Cross-reactive protection against influenza A virus infections by an NS1-specific CTL clone.

An influenza A subtype cross-reactive CTL clone (A-11) was established following stimulation of A/PR/8 virus-immune spleen cells of Balb/C (H-2d) mice. This T cell clone lysed target cells infected with influenza viruses of the H1, H2, or H3 subtypes, and recognizes a conserved epitope on the NS1 protein. The clone is restricted by the H-2Ld allele. Adoptive transfer of A-11 significantly reduced virus titers in the lungs of mice infected with influenza A viruses of the H1, H2, or H3 subtypes. These results suggest that the conserved epitope on NS1 which is recognized by A-11 may be a useful component to consider for inclusion in experimental cross-reactive influenza 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.     

Priming of virus-immune memory T cells in newborn mice.

Neonatal BALB/c mice can be primed at birth by intravenous inoculation of a small dose of A/Puerto Rico/8/34 (H1N1) (PR8) influenza virus. UV-inactivated PR8 virus, or PR8 virus complexed with monoclonal antibody to give a secondary cytotoxic T lymphocyte response when restimulated in vitro as adults. The frequency of responding T cells after secondary stimulation in vitro is approximately 40% of that found for adult mice primed intraperitoneally with a large dose of PR8 virus. The majority of the T cells generated from mice primed at birth or as adults are cross-reactive for H-2-compatible targets infected with the PR8 (H1N1) or A/Hong Kong/X31 (H3N2) viruses. Splenocytes from neonates receiving UV-inactivated vaccinia virus at birth give an augmented secondary cytotoxic T lymphocyte response when restimulated 8 days later in adoptive irradiated adult hosts. We found no indications of specific immunological unresponsiveness in mice exposed to either virus.     

Recognition of disparate HA and NS1 peptides by an H-2Kd-restricted, influenza specific CTL clone.

CTLs (CD8+) are known to recognize exogenous peptide in the context of class I MHC molecules. We observed that an influenza subtype H1 and H2 cross-reactive CTL clone B7, which was stimulated by a fusion protein containing a portion of HA2 subunit of A/PR/8 virus HA, recognized a synthetic peptide (residues 515-526) of the HA2 subunit of A/PR/8 virus strain. This CTL clone also recognized a structurally disparate NS1 peptide 50-68 of the same A/PR/8 virus. We examined the recognition of the NS1 peptide 50-68 and the HA peptide 515-526 by the subcloned CTL clone, B7-B7. Cold target inhibition experiments showed that the recognition of the HA peptide by the CTL clone B7-B7 could be competed by NS1 peptide-treated target cells and vice versa. The recognition of both NS1 peptide and HA peptide by the CTL clone B7-B7 was restricted by the same allele, H2Kd. In addition, this NS1 peptide requires approximately a 600-fold higher concn for optimal CTL recognition than did the HA peptide. We conclude that the TCR on clone B7-B7 recognizes the HA peptide or the NS1 peptide as comparable complexes with the same class I MHC molecules, although there is no obvious homology in the primary sequences of HA 515-526 and NS1 50-68 peptides. CTLs elicited with certain antigens appear to recognize distinctively different antigens complexed to the same presenting MHC molecule.     

Cross-reactive protection against influenza A virus by a topically applied DNA vaccine encoding M gene with adjuvant

The skin is rich with immunocompetent cells and therefore immunization through the skin is an attractive alternative to the invasive vaccination methods currently used. In this study the backs of mice were gently shaved, hydrated, and painted with a DNA vaccine encoding influenza M protein with adjuvant. The immunized mice were then challenged with two mouse-adapted strains of the influenza virus A: A/PR/8/34 (H1N1) and A/Udorn/72 (H3N2). This adjuvanated and topically applied DNA vaccine efficiently induced cytotoxic as well as humoral immune response and provide cross-reactive protection against several strains of influenza A virus. For better protection against virus infection, it will be necessary to select and combine the DNA vaccine with an appropriate adjuvant.     

High-yield reassortant influenza vaccine production virus has a mutation at an HLA-A 2.1-restricted CD8+ CTL epitope on the NS1 protein.

Current influenza virus vaccines are prepared using high-yield reassortant virus strains obtained from a mixed infection of the new virus strain and a prototype high-yielding virus strain. The high-titered reassortant virus strain used as vaccine seed virus possesses the recent virus HA and NA and contains the internal genes from the high-growing prototype parent. We established a human CD8(+) cytotoxic T cell (CTL) line, 10-2C2, which recognizes an HLA-A2.1-restricted influenza A virus H1, H2, H3 cross-reactive T cell epitope on amino acids 122-130 of the NS1 protein, and unexpectedly we observed that there was decreased lysis of target cells infected with the A/Texas/36/91 (H1N1) vaccine virus strain compared to the lysis of target cells infected with the prototype A/PR/8/34 (H1N1) virus. RT-PCR results showed that the A/Texas vaccine virus strain contained a quasispecies. Approximately 50% of viral RNA of the NS1 gene had a nucleotide substitution that resulted in the N --> K amino acid change at the sixth position of the nonamer peptide. Current influenza vaccines are inactivated and do not contain the NS1 protein; however, future influenza vaccines may include live attenuated vaccines and with this mutation a live virus would fail to induce a CD8(+) CTL response to this epitope in individuals with HLA-A2.1, a very common allele, and potentially have reduced efficacy.     

Cytotoxic lymphocytes and antibody-dependent complement-mediated cytotoxicity induced by administration of influenza vaccine.

Recently defined aspects of cellular and humoral antiviral immunity were evaluated in 10 young adults given influenza vaccines containing A/USSR/77 (H1N1) antigens. Cytotoxic lymphocytes were measured by using cryopreserved lymphocytes as effector cells and syngeneic, virus-infected lymphocytes as target cells. An assay previously developed in this laboratory was adapted for the measurement of antibody-dependent, complement-mediated cytotoxicity. Antiviral cytotoxic lymphocyte responses were detected in 5 of 10 volunteers between 3 and 10 days after the initial vaccination. These responses were found both in individuals who were previously primed and in individuals who were not primed to influenza A/USSR/77 antigens. The complement-mediated lysis assay was found to be more sensitive than the hemagglutination inhibition test and probably detected antibodies to both subtype-specific and cross-reactive antigenic determinants. These responses to influenza antigens are similar to those obtained in studies of murine influenza which indicate that cytotoxic lymphocytes and antibody-dependent, complement-mediated cytotoxicity have a role in the early response to acute infection.     

Viral recognition by influenza A virus cross-reactive cytotoxic T (Tc) cells: the proportion of Tc cells that recognize nucleoprotein varies between individual mice

L cells (H-2k) transfected with DNA coding for the A/NT/60/68 influenza nucleoprotein (NP) gene have been found previously to provide a target cell for cytotoxic T (Tc) cells. In this report we have studied the repertoire of cytotoxic memory cells in mice of different strains primed by infection with several influenza A subtypes. L cells transfected with NP and Db genes enabled us to estimate the proportion of A virus cross-reactive Tc cells specific for NP. The most striking feature of our results was variation in the frequency of NP-specific Tc cells between individuals of an inbred mouse strain. Occasional individuals, although showing a strong A virus cross-reactive Tc response, had no A virus cross-reactive Tc cells that recognized NP. The NP-directed Tc repertoire represented between 0-40% of lysis of target cells infected with a heterologous type A virus. Thus, a significant proportion of A virus cross-reactive Tc cells must have different specificities for type A influenza virus. In C3H-H-2o2 mice, Dk is a low responder allele for NP recognition by Tc cells.     

Recruitment,activation and proliferation of CD8+ memory T cells in an immunoprivileged site

The capacity of a memory cytotoxic T lymphocyte (CTL) population to protect against viral infections is well established, but the processes underlying this protection are less well understood. We have used heterotypic intranasal immunization with influenza A/X31 (H3N2) to protect against a subsequent infection with the neurovirulent influenza A/WSN (H1N1) in either the cerebrospinal fluid or the immunoprivileged brain parenchyma. Viral clearance from both sites was associated with a local infiltration and proliferation of A/WSN-specific CD8⁺ T cells. Infection in the cerebrospinal fluid elicited a proliferative response in the draining lymph nodes, an anti-H1N1 serum antibody response and an increase in the extracerebral A/WSN-specific CTL precursor frequency. In contrast, infection in the brain parenchyma elicited no lymph node proliferative response or serum antibody response and caused a transient decrease in the extracerebral CTL precursor frequency. Thus the memory CTL population protected against an intracerebral viral infection independent of any immune response occurring in systemic lymphoid tissue.     

穿孔素介导的细胞凋亡机制在抗流感病毒感染免疫防御中作用的研究

目的：穿孔素介导的细胞凋亡机制在流感病毒初次感染中作用的研究。方法：用流感病毒A／PR／8／34经鼻感染穿孔素基因敲除鼠和同源对照C57BL／6小鼠，采用PFU方法测定肺内流感病毒增殖状况；免疫组织化学染色方法观察小鼠病毒感染后感染细胞的凋亡情况；利用乳酸脱氢酶释放法检测感染鼠脾淋巴细胞NK活性及CTL杀伤活性。结果：穿孔素基因缺乏导致流感病毒在小鼠肺内大量增殖；小鼠清除感染病毒所需时间延长；病毒感染细胞发生凋亡的时间亦因穿孔素的缺乏而延迟；感染小鼠脾淋巴细胞NK活性及CTL杀伤活性均显著降低。结论：穿孔素依赖的细胞介导的细胞毒效应在控制流感病毒初次感染，快速清除感染病毒方面起重要作用。     

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.     

Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response

Regulation of the expression of influenza A/PR/8/34 hemagglutinin (HA) by the vaccinia virus promoters PF (early), P7.5 (early and late) and PL11 (late) has been demonstrated using HA-vaccinia recombinant viruses VV-PR8-HA3, VV-PR8-HA6 and VV-PR8-HA, respectively. Levels of HA on the surface of VV-PR8-HA3 (PF)-infected cells were lower than with either VV-PR8-HA6 (P7.5) or VV-PR8-HA8 (PL11). Expression of HA under the control of the late promoter PL11 was inhibited in the absence of DNA replication. All three recombinant viruses stimulated a specific antibody response in mice which was dependent on the presence of infectious virus. Recognition of HA by cytotoxic T lymphocytes (CTL) was assessed by the ability of the viruses to stimulate naive precursors in vivo, to restimulate primed CTL in vitro and by target cell recognition. HA expressed under the control of either of the promoters with early function (PF or P7.5) was recognized by CTL when VV-PR8-HA3 or VV-PR8-HA6 were used to prime or restimulate splenocytes or to infect target cells. On the other hand, HA expressed by VV-PR8-HA8 (PL11) failed to prime for a CTL response in naive CBA/H mice, was ineffective at restimulation of primed splenocytes and failed to produce target cells for recognition by specific CTL. However, in BALB/c mice VV-PR8-HA8 did prime for a specific CTL response. These studies show that HA synthesized early in infection was recognized by both B and T cells while HA expressed after DNA replication was not generally recognized by T cells. The implications of the observations with the late promoter with respect to the use of late promoters in potential vaccinia virus-based vaccines are considered.     

Local IL-4 expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic T cell responses

We studied the effect of lung-specific IL-4 expression on the T cell response during primary and secondary heterologous infection with influenza virus by using transgenic mice that express IL-4 under a lung-specific promoter. Subsequent to primary infection with a type A/H1N1 influenza virus these transgenic mice exhibited similar local recruitment of CD4(+) and CD8(+) T cells and only slightly decreased virus-specific CTL activity. However, during secondary challenge with a heterologous influenza virus, the local infiltration with virus-specific, MHC class I-restricted CD8(+) T cells was significantly decreased compared to that of nontransgenic littermates. The ability of IL-4 transgenic mice to clear the heterologous infection was delayed but not abrogated. This was associated with a faster virus-neutralizing antibody response in IL-4 transgenic mice and with their ability to mount significant Th1 responses even in the presence of increased local IL-4 expression. Our observations demonstrate a negative regulatory effect of IL-4 on memory Tc1/CD8(+) T cells, but are also consistent with complementary mechanisms important for virus clearance such as virus-neutralizing antibodies. The reduction of memory CTL in the presence of IL-4 might have consequences for understanding the course of influenza infection in situations where T(H)2 immunity is increased.     

Effect of influenza virus on the immune responsiveness of animals.

Infection of mice with A/Hong Kong/1/68 (H3N2) and A/PR8/34 (H0N1) influenza virus strains resulted in a significant inhibition of the formation of antibody-producing cells in response to administration of sheep erythrocytes and a reduced capacity of spleen cells to induce "graft-versus-host" reaction. The immunosuppression caused by influenza infection was observed for a long period of time post infection (3--6 months). The extent of inhibition of antibody production depended on the dose of virus, route of inoculation, the sequence of infection and immunization and the internal between them. Heat-inactivated virus exerted no immunosuppressive effect.     

Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity

Mice previously infected with an aerosol of A/Rec 31 influenza virus were strongly protected against an aerosol challenge with A/Vic influenza as judged by lung virus titers recovered 2 days after the challenge infection. Such complete homotypic immunity was not achieved by priming with live Rec 31 virus injected i.v. or UV-inactivated Rec 31 virus administered s.c. together with Al(OH)₃ and saponin. The reason for the superior protective effect of the natural infection was investigated. The protection induced by respiratory infection with Rec 31 virus was specific for influenza A viruses. It was not correlated with specific serum hemagglutination inhibition antibody titer or cross-reactive cytotoxic T (T_c) cell reactivity. Moreover, the transfer of splenic and lymphoid T cell populations with strong secondary T_c activity did not significantly reduce lung virus titers in recipient mice 3 days after infection. The protection however occurred in parallel with the presence of cross-reactive IgA antibody in the lung washings. It thus appears that local secretory IgA plays a causal role in the prevention of cross-infection by influenza A virus. Serum antibody and T_c cells, on the other hand, may be crucial for recovery from such infection. All mice primed with live Rec 31 virus, administered i.v. or by aerosol and expressing equally high levels of T_c reactivity, survived a lethal challenge with A/PR8 virus. The same challenge, however, killed half of the mice immunized s.c. with inactivated Rec 31 virus which induced only a low level of T_c reactivity.     

Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor beta chain marker

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.     

Reproductive activity of the influenza A virus in the splenocytes of experimental animals with mono- and mixed infections

Virus-induced processes in organs and tissues of Syrian hamsters in relation to the influenza A virus strain used (HON1 or H3N2), age of the animals, and in the presence of mixed infection were compared. The infection of young hamsters with A/PR8/34 and A/Bangkok/1/79 viruses was shown to induce the synthesis of viral proteins NP and M in spleen cells lasting for up to 15 days (the observation period). In mixed influenza and respiratory syncytial virus infection the possibility of influenza virus genome expression did not change. After infection of mature hamsters, synthesis of virus-specific NP and M proteins in splenocytes was observed only in the animals infected with influenza A/PR8/34 virus but not in those infected with the less pathogenic influenza A/Bangkok/1/79 virus.     

Increased immunogenicity of inactivated influenza virus vaccine containing purified surface antigen compared with whole virus in elderly women.

Thirty-eight elderly female subjects (aged 80 +/- 7 years, mean +/- standard deviation) were randomized to immunization with trivalent inactivated influenza virus vaccine containing either purified surface antigen (n = 18) or whole virus (n = 20) components from A/Texas/36/91 (H1N1), A/Beijing/353/89 (H3N2), and B/Panama/45/90 strains. Humoral and cellular immune responses were assessed by measuring serum hemagglutination inhibition antibodies and cytotoxic T lymphocyte (CTL) activity at 0 and 3 weeks postvaccination. Serological responses to both of the type A vaccine strains following immunization with surface antigen vaccine (SAV) were significantly more frequent and greater in magnitude than those induced by whole-virus vaccine. Antibody responses to the B/Panama component were modest and did not differ significantly between the two vaccines. Persons given SAV, but not those given whole-virus vaccine, had a small ¿ but significant increase in mean percent specific lysis of influenza A (H1N1) virus-infected autologous targets by peripheral blood mononuclear cells which were stimulated in vitro with influenza A (H1N1) virus. The H1N1-stimulated cytotoxic effectors induced by SAV were CD8+ and were not cross-reactive against H3N2-infected targets. Influenza B virus-specific CTL responses were not observed with either vaccine. These results suggest that currently available subunit influenza virus vaccines may offer an advantage over inactivated whole-virus preparations for inducing humoral and cellular immune responses in the elderly, although the CTL response may be too limited to be of physiological significance.