Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.     

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.     

The analysis of the monoclonal immune response to influenza virus. III. The relationship between stimulation of virus-primed precursor B cells by heterologous viruses and reactivity of secreted antibodies.

Individual splenic precursor B cells from BALB/c mice primed with influenza virus PR8[A/PR/8/34 (H0N1)] were stimulated in vitro in the splenic fragment culture system by homologous or various heterologous influenza viruses. The specificity of the stimulated precursor cells was determined by analysis of the antibodies secreted by the ensuing plasma cell clone in a radioimmunoassay (RIA). Viruses of the H2N2 and H3N2 subtypes were unable to stimulate hemagglutinin (HA)- or neuraminidase (NA)-committed precursor B cells but did efficiently stimulate chicken host component (ChHC)-committed precursors. Viruses of the H1N1 and H0N1 subtypes could stimulate precursors committed to any of the three viral surface components. Analysis of the fine specificity of HA-committed B cells showed that BEL(H0N1) and CAM(H1N1) stimulated almost exclusively precursors whose clonal antibody product reacted with the stimulating virus in the RIA. On the other hand, WSE and MEL (both H0N1) quite frequently were able to stimulate precursors whose clonal antibody product did not react with the stimulating virus in the RIA. These results suggest that the stimulatory interaction of viruses with the cell-bound immunoglobulin receptors is slightly less affinity dependent than the antigen-antibody interaction in the RIA.     

T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.     

In vitro antibody response to influenza virus. II. Specificity of helper T cell recognizing hemagglutinin

Intraperitoneal immunization of mice with liver influenza virus was shown to induce helper T (TH) cells with specificity for the hemagglutinin (HA). The interaction of virus-primed TH cells with purified HA was studied independently of B cell reactivity to the same antigen by using the generation of nonspecific help as an index of activation of HA-specific TH cells. TH cells from mice primed with any of the H3 viruses A/Aichi/68 X A/Bel/42 (H3N1), A/Memphis/102/72 X A/Bel/42 (H3N1) or A/Port Chalmers/73 (H3N2) were strongly cross-reactive towards HA of other strains within the H3 subtype. In addition, several examples of cross-reactivity towards HA of a different subtype were observed, usually of a lower magnitude. TH cells from mice primed to any of the H3 viruses above or to A/Bel/42 (H1N1) virus cross-reacted with the HA of A/Japan/305/57 (H2); furthermore, priming with A/Bel/42 or with A/Jap/305/57 X A/Bel/42 (h2N1) virus yielded TH cells that cross-reacted with certain of the H3 HA preparations. The cross-reactivity observed between subtypes was not due to the common chicken host carbohydrate component of HA, since no response to the purified type A HA preparations was obtained with T cells from mice primed with egg-grown influenza B/Hong-Kong/8/73 virus. The results indicate that HA of different subtypes may share cross-reactive antigenic determinants recognized by TH cells. Within a subtype, HA are highly cross-reactive with respect to tH cell recognition.     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.     

Healthy human subjects have CD4+ T cells directed against H5N1 influenza virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.     

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.     

Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells.

The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.     

Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin- or neuraminidase-expressing DNA in BALB/c mice

Avian influenza viruses of H9N2 subtype are widely spread in avian species. The viruses have recently been transmitted to mammalian species, including humans, accelerating the efforts to devise protective strategies against them. In this study, an avian influenza H9N2 virus strain (A/Chicken/Jiangsu/7/2002), isolated in Jiangsu Province, China, was used to infect BALB/c mice for adaptation. After five lung-to-lung passages, the virus was stably proliferated in a large quantity in the murine lung and caused the deaths of mice. In addition, we explored the protection induced by H9N2 virus hemagglutinin (HA)- and neuraminidase (NA)-expressing DNAs in BALB/c mice. Female BALB/c mice aged 6-8 weeks were immunized once or twice at a 3-week interval with HA-DNA and NA-DNA by electroporation, respectively, each at a dose of 3, 10 or 30microg. The mice were challenged with a lethal dose (40x LD(50)) of influenza H9N2 virus four weeks after immunization once or one week after immunization twice. The protections of DNA vaccines were evaluated by the serum antibody titers, residual lung virus titers, and survival rates of the mice. The result showed that immunization once with not less than 10microg or twice with 3microg HA-DNA or NA-DNA provided effective protection against homologous avian influenza H9N2 virus.     

Subtype cross-reactive, infection-enhancing antibody responses to influenza A viruses.

Antibody-dependent enhancement of the uptake of influenza A virus by Fc receptor-bearing cells was analyzed by using virus strains of the three human influenza A virus subtypes, A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and A/Port Chalmers/1/73 (H3N2). Immune sera obtained from mice following primary infection with an H1N1, H2N2, or H3N2 subtype virus neutralized only virus of the same subtype; however, immune sera augmented the uptake of virus across subtypes. Immune sera from H1N1-infected mice augmented uptake of the homologous (H1N1) and H2N2 viruses. Antisera to the H2N2 virus augmented the uptake of virus of all subtypes (H1N1, H2N2, or H3N2). Antisera to the H3N2 virus augmented the uptake of the homologous (H3N2) and H2N2 viruses. These results show that subtype cross-reactive, nonneutralizing antibodies augment the uptake of influenza A virus strains of different subtypes. Antibodies to neuraminidase may contribute to the enhanced uptake of viruses of a different subtype, because N2-specific monoclonal antibodies augmented the uptake of both A/Japan/305/57 (H2N2) and A/Port Chalmers/1/73 (H3N2) viruses.     

In vitro antibody response to influenza virus. I. T cell dependence of secondary response to hemagglutinin.

A good secondary IgG response to the hemagglutinin (HA) of influenza virus has been obtained in vitro in Marbrook-type cultures of influenza-primed mouse spleen cell suspensions stimulated with inactivated influenza virus. Anti-HA antibody was quantitated by a solid phase radioimmunoassay (RIA) by using purified HA as substrate. The T dependence of this secondary response was shown by depletion of T cells and reconstitution with a source of primed or unprimed T cells. The help given by T cells primed to the homologous virus was many times greater than that given by unprimed T cells, although the latter was significant. The system described will allow investigation of the specificity requirements of helper T cells engaged in the anti-HA response.     

Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. II. Sequential infection of mice simulates human experience

In man, vaccination with neuraminidase (NA) in H7N2 virus hybrids elicits greater anti-NA response than does N2 NA in H3N2 conventional vaccine, presumably because humans are H3 hemagglutinin (HA) primed and anti-H3 anamnestic response depresses concomitant N2 responses by antigenic competition. In a laboratory model, BALB/c mice were primed by different schedules of infection with H3N1, H3N2, and H3N7 viruses and given H3N2 and H7N2 vaccines equivalent in NA immunogenicity. In schedules using sequential infections, but not after a single infection with any virus, anti-N2 booster response was fourfold greater with H7N2 vaccine and was reciprocal to the magnitude of anti-H3 response. Thus, HA-influenced suppression of immunologic response to viral NA requires adequate HA priming but is not unique to man and can be studied in the murine model. An incidental finding of this study was the sharing of cross-reactive determinants by N1, N2, and N7 NA.     

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection.

BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.     

MyD88 signaling is indispensable for primary influenza A virus infection but dispensable for secondary infection

Recent studies have revealed that innate immunity is involved in the development of adaptive immune responses; however, its role in protection is not clear. In order to elucidate the exact role of Toll-like receptor (TLR) or RIG-I-like receptor (RLR) signaling on immunogenicity and protective efficacy against influenza A virus infection (A/PR/8/34 [PR8]; H1N1), we adapted several innate signal-deficient mice (e.g., TRIF(-/-), MyD88(-/-), MyD88(-/-) TRIF(-/-), TLR3(-/-) TLR7(-/-), and IPS-1(-/-)). In this study, we found that MyD88 signaling was required for recruitment of CD11b(+) granulocytes, production of early inflammatory cytokines, optimal proliferation of CD4 T cells, and production of Th1 cytokines by T cells. However, PR8 virus-specific IgG and IgA antibody levels in both systemic and mucosal compartments were normal in TLR- and RLR-deficient mice. To further assess the susceptibility of these mice to influenza virus infection, protective efficacy was determined after primary or secondary lethal challenge. We found that MyD88(-/-) and MyD88(-/-) TRIF(-/-) mice were more susceptible to primary influenza virus infection than the B6 mice but were fully protected against homologous (H1N1) and heterosubtypic (H5N2) secondary infection when primed with a nonlethal dose of PR8 virus. Taken together, these results show that MyD88 signaling plays an important role for resisting primary influenza virus infection but is dispensable for protection against a secondary lethal challenge.     

Inhibition of influenza A virus hemagglutinin and induction of interferon by synthetic sialylated glycoconjugates.

Multivalent forms of neoglycoproteins and polyacrylamides containing sialic acid were prepared and shown to be potent inhibitors of influenza A virus (H3N2) hemagglutinin with chick red blood cells. The synthetic sialylated glycoconjugates, although they were neuraminidase substrates, did not suppress viral neuraminidase and did not reduce infectivities in chick embryos. The copolyacrylamide conjugate containing a spacer group of approximately 11 A (1 A = 0.1 nm) between the polymer backbone and the sialic acid residues was the best hemagglutinin inhibitor. Moreover, it exhibited promising interferon-inducing properties.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

Various approaches to designing the functionally active determinants as illustrated by the influenza virus A(H3N2) hemagglutinin]

Hexapeptide Lys-Gly-Pro-Asp-Ser-Gly analogous to the immunodominant fragment 141-146 of the epitope A of the influenza virus A(H3N2) hemagglutinin is synthesized. Conjugated with thyroglobulin and hemocyanine, the hexapeptide induced formation of highly specific antibodies with heterolytic properties in CBA mice. Antihexapeptide antibodies interact not only with the homologous antigen but also with hemagglutinin and influenza virus. Choice of the hexapeptide sequence is discussed.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Cationic lipid/DNA complex-adjuvanted influenza A virus vaccination induces robust cross-protective immunity

Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.