Induction of interferon alpha from human lymphocytes by autologous, dengue virus-infected monocytes.

Human monocytes actively replicate dengue virus. To dissect the primary immune responses to dengue virus-infected monocytes (DV-monocytes), we analyzed the interaction between autologous DV-monocytes and the peripheral blood lymphocytes (PBL) of dengue nonimmune donors. Interferon (IFN) activity was detected when PBL were cultured with DV-monocytes. Cell contact between PBL and DV-monocytes was required for IFN production; however, MHC compatibility between PBL and monocytes was not necessary. DV-monocytes fixed with paraformaldehyde or glutaraldehyde, which produced no infectious virus, also induced high levels of IFN from PBL. The ability of DV-monocytes to induce IFN correlated with the appearance of dengue antigens. The PBL that produce IFN were characterized by FACS sorting using monoclonal and polyclonal antibodies. HLA-DR+ and T3- cells produced high titers of IFN, while HLA-DR- and T3+ cells produced very low or undetectable levels of IFN. Moderate titers of IFN were produced by cells contained in B cell fractions (surface immunoglobulin-positive, B1+, and Leu-12+), and cells contained in natural killer cell fractions (Leu-11+ and OKM1+). Therefore, IFN-producing cells are heterogeneous, and the predominant producer cells are characterized as HLA-DR+ and non-T lymphocytes. The IFN produced was characterized by RIA using mAbs to IFN-alpha and IFN-gamma. The IFN-alpha was the predominant IFN produced; in addition, a low level of IFN-gamma was also detected in some experiments. The culture fluids obtained from PBL exposed to autologous DV-monocytes, which contained high IFN activity, completely inhibited dengue virus infection of monocytes. These results suggest that IFN-alpha produced by PBL exposed to DV-monocytes may play an important role in controlling primary dengue virus infection.

Immune interferon produced to high levels by antigenic stimulation of human lymphocytes with influenza virus.

Influenza virus stimulation of human lymphocytes induced high levels of immune interferon in lymphocyte cultures. The lymphocytes of normal adults produced approximately 1,000 U/10(6) cells, which was in large part gamma interferon. The lymphocytes of individuals recently vaccinated yielded very high levels (10-50,000 U/10(6) cells) of interferon. The interferon was pH 2 labile, and was not neutralized by antisera to alpha or beta interferon. It did not bind to a monoclonal antibody to alpha interferon, and after partial purification it had characteristics identical to human gamma interferon induced by phytohemagglutinin. The highest yields were produced by treatment of stimulator cells with live virus. Stimulation by whole inactivated virus resulted in lower levels of interferon, and purified hemagglutinin did not induce interferon. The antigen responsible for stimulating the lymphocyte response and interferon induction is a cross-reactive determinant present on all human and non-human influenza viruses tested.

Influenza virus subtype-specific cytotoxic T lymphocytes lyse target cells coated with a protein produced in E. coli.

We have tested the ability of the c13 protein, which is a hybrid protein of the first 81 amino acids of the viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutination produced in E. coli, to render target cells susceptible to the lytic activity of influenza virus-specific cytotoxic T lymphocytes (CTL). The results showed that P815 cells coated with c13 protein were lysed by PR8 virus-induced secondary CTL derived from BALB/c mice. Cold-target inhibition tests clearly demonstrated that c13 protein-coated P815 cells were recognized by an H1 subtype-specific CTL population. Furthermore, PR8 virus-induced CTL derived from C3H mice did not lyse c13 protein-coated P815 cells, suggesting that c13 protein was recognized by CTL in conjunction with H-2d products. These findings suggest that this protein interacts with the cellular plasma membrane and makes target cells recognizable by H-2-restricted, influenza virus subtype-specific CTL.

Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity.

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN-gamma after stimulation with dengue 3 antigens, and also produced IFN-gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus-infected autologous LCL, and they did not lyse uninfected autologous LCL. The lysis of dengue antigen-pulsed or virus-infected LCL by the crossreactive CTL clones that we have examined is restricted by HLA DP or DQ antigens. These results indicate that primary dengue virus infections induce predominantly crossreactive memory CD4+ T lymphocytes. These crossreactive T lymphocytes proliferate and produce IFN-gamma after stimulation with a virus strain of another serotype, and demonstrate crossreactive cyotoxic activity against autologous cells infected with heterologous dengue viruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Active immunization against virus infections due to antigenic drift by induction of crossreactive cytotoxic T lymphocytes.

We have examined whether active immunization with c13 protein, a hybrid protein of the first 81 amino acids of the viral NS1 nonstructural protein and the HA2 subunit of A/PR/8 (H1N1) hemagglutinin, could protect BALB/c mice from challenge with A/PR/8 H1 subtype virus. Mice immunized with the c13 protein had a significant reduction of pulmonary virus titers with A/PR/8 (H1) virus, but failed to limit the replication of A/PC (H3) virus, which reflects the in vitro CTL activity of c13 immune spleen cells. We observed that the epitope recognized by HA2 specific CTL, which are induced by a derivative of c13 protein, is highly conserved among H1 and H2 subtype virus strains. This led us to test whether active immunization with c13 protein would also limit pulmonary virus replication in mice infected with the A/TW virus, a virus of the H1 subtype, which was isolated in 1986, and with a virus of the H2 subtype, A/Japan/305/57. Immunized mice had significantly lower lung virus titers than did control mice, and did not possess any neutralizing antibodies to the challenger viruses. These results indicate that active immunization with a fusion protein containing the cross-reactive CTL epitope protects mice from influenza infection by inducing CTL against influenza A H1 and H2 subtype virus strains, which markedly vary in their antibody binding sites on the HA1. The ability to induce active cross-reactive immunization with a fusion protein which contains a highly conserved CTL epitope offers a model for vaccine approaches against viruses which undergo significant variations in their antibody binding sites.

Hemagglutinin-specific cytotoxic T-cell response during influenza infection.

Specific cytotoxic thymus-derived (T) lymphocytes were detected in the cervical lymph nodes and spleen during influenza infection of mice. The cytotoxic T cells can distinguish target cells infected with different influenza A subtypes. Infection with parent viruses and their recombinant progeny possessing the hemagglutinin of one parent and the neuraminidase of the other demonstrated that significant cytotoxicity occurred only when the hemagglutinin of the immunizing viruses was the same as that of the virus used to infect the target cell. In addition to this specific cytotoxic response to the major surface antigen, a cross-reactive response could be detected when the relatively nonpermissive L cell was used as the target cell. These results indicate there is a specific cytotoxic T-cell response to the surface hemagglutinin, and a cross-reactive cytotoxic response, not directed to the hemagglutinin, during influenza infection. The cytotoxic T-cell response specific for the hemagglutinin antigen may play an important role in in vivo immunity to influenza.

Evidence that cytotoxic T cells are part of the host's response to influenza pneumonia.

Cytotoxic T cells were detected in the cervical lymph nodes, lungs, spleen, and peripheral blood of mice with influenza. Lymphocytes decreased in the peripheral circulation and increased in the lung during the period of acute inflammation and pneumonia. Peak cytotoxic T-cell activity was present at the time of marked pulmonary infiltration, and it decreased with resolution of the pneumonia. The cytotoxic T cells in the lung were shown to be H-2 restricted and specific for the hemagglutinin of the infecting virus. The results indicate that hemagglutinin specific cytotoxic T cells are (a) induced during influenza infection; (b) they circulate in the blood; (c) they are present in greatest number; and (d) they have their peak cytotoxic effect when pneumonia is most marked. We interpret the results to indicate that specific cytotoxic T cells in the infected target organ are part of the immunological and pathological response to virus infection.

Hemagglutinin-specific complement-dependent cytolytic antibody response to influenza infection.

The host defense response to influenza infection is complex. Specific humoral antibodies develop to the strain-specific surface antigens, the hemagglutinin and the neuraminidase, and to the internal antigens (matrix and nucleoprotein) which are common to all influenza A viruses (1). Antibodies to the hemagglutinin, which is the major surface antigen, neutralize viral infectivity (2). In addition to antibodies which have been detected against virion antigens, a cytotoxic T-cell response with specificity against the viral hemagglutinin on influenza-infected target cells (3-5) has been recently described. A more cross-reactive cytotoxic T-cell response has also been observed when a nonpermissively infected target cell is used in cytotoxicity assays (6,7). The present report describes the development during influenza infection and after vaccination of a cytolytic humoral antibody response which is directed against the hemagglutinin on infected target cells. This antibody-mediated lysis of infected cells in complement dependent, as has been reported with other virus infections (8-11).

Influenza virus hemagglutinin-specific cytotoxic T cell response induced by polypeptide produced in Escherichia coli.

We have tested the abilities of various polypeptides of A/PR/8/34 (H1N1) virus, constructed by recombinant DNA techniques, to induce influenza virus-specific secondary cytotoxic T lymphocyte (CTL) responses. A hybrid protein (c13 protein), consisting of the first 81 amino acids of viral nonstructural protein (NS1) and the HA2 subunit of viral hemagglutinin (HA), induced H-2-restricted, influenza virus subtype-specific secondary CTL in vitro, although other peptides did not. Using a recombinant virus, the viral determinant responsible for recognition was mapped to the HA2 portion of c13 protein. Immunization of mice with c13 protein induced the generation of memory CTL in vivo. The CTL precursor frequencies of A/PR/8/34 virus- and c13 protein-immune mice were estimated as one in 8,047 and 50,312, respectively. These results indicate that c13 protein primed recipient mice, even though the level of precursor frequency was below that observed in virus-immune mice.

A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities.

It is generally accepted that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus-specific CD8+ CTLs of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships between the serotype specificities of T cells and the amino acid sequence of the recognized peptides, we examined the ability of this viral peptide D4.NS3.500-508 (TPEGIIPTL) to stimulate T lymphocytes of an HLA-B35-positive, dengue virus type 4-immune donor. Peptide stimulation of the PBMC generated dengue virus-specific, HLA-B-35-restricted CD8+ CTL clones. These clones lysed dengue virus-infected autologous cells, as well as autologous target cells pulsed with this peptide. Four patterns of dengue virus serotype specificities were demonstrated on target cells infected with dengue-vaccinia recombinant viruses or pulsed with synthetic peptides corresponding to amino acid sequences of four dengue virus serotypes. Two serotype-specific clones recognized only D4V. Three dengue virus subcomplex-specific clones recognized D1V, D3V, and D4V, and one subcomplex-specific clone recognized D2V and D4V. Three dengue virus serotype-cross-reactive clones recognized D1V-D4V. Thus, a single nine-amino acid peptide induces proliferation of a heterogeneous panel of dengue virus-specific CD8+ CTL clones that are all restricted by HLA-B35 but have a variety of serotype specificities. Peptides that contain a single amino acid substitution at each position of D4.NS3.500-508 were recognized differently by the T cell clones. These results indicate that a single epitope can be recognized by multiple CD8+ CTLs that have a variety of serotype specificities, but the manner of recognition by these multiple CTLs is heterogeneous.

Antibody-enhanced infection by HIV-1 via Fc receptor-mediated entry.

Monocytes and macrophages, which may play a central role in the pathogenesis of infection with human immunodeficiency virus type 1 (HIV-1), express the CD4 molecule and Fc receptors (FcR) for immunoglobulin G (IgG). To explore the possibility that FcR mediate HIV-1 infection of monocytes, studies were conducted with the human monocytic cell line U937. These cells were exposed to HIV-1 complexed with various concentrations of serum from HIV-1 antibody-positive individuals and monitored for HIV-1 replication. Serum samples from antibody-negative normal individuals did not affect virus yields. High concentrations of antibody-positive sera showed virus-neutralizing activity; however, cells infected with HIV-1 in the presence of antibody-positive sera at subneutralizing concentrations significantly enhanced virus replication. This infection enhancement was blocked by heat-aggregated gamma-globulin. Moreover, the IgG fraction from an HIV-1 antibody-positive serum enhanced HIV-1 infection at the same serum dilution equivalents. In contrast, IgG-F(ab')2 did not enhance HIV-1 infection but showed neutralizing activity with HIV-1. These results are compatible with the concept of FcR-mediated infection enhancement and suggest that this immunological response to HIV-1, instead of protecting the host, potentially facilitates the infection.

Subacute sclerosing panencephalitis: experimental infection in primates.

Measles virus isolated from the brain of a 12-year-old boy with subacute sclerosing panencephalitis caused a chronic, progressive encephalitis in experimentally infected rhesus monkeys. The infection was eventually fatal in spite of pre-existing measles immunity and a vigorous secondary antibody response in serum and cerebrospinal fluid of the infected animals. The findings provide a basis for studies into the pathogenesis and possible treatment of the human disease.

Human T cell responses to dengue virus antigens. Proliferative responses and interferon gamma production.

The severe complications of dengue virus infections, hemorrhagic manifestations and shock, are more commonly observed during secondary dengue virus infections than during primary infections. It has been speculated that these complications are mediated by cross-reactive host-immune responses. We have begun to analyze human T cell responses to dengue antigens in vitro to explain the possible role of T lymphocytes in the pathogenesis of these complications. Dengue antigens induce proliferative responses of PBMC from dengue antibody-positive donors, but do not induce specific proliferative responses of PBMC from dengue antibody-negative donors. IFN gamma is detected in the culture fluids of dengue-immune PBMC stimulated with dengue antigens. The cells that proliferate in the dengue antigen-stimulated bulk cultures have CD3+, CD4+, CD8-, CD16-, and CD20- phenotypes. Dengue-specific T cell lines were established using limiting dilution techniques. They have CD3+, CD4+, and CD8- phenotypes, and produce IFN gamma in response to dengue antigens. Culture fluids from dengue-immune PBMC stimulated with dengue antigens, which contain IFN gamma, augment dengue virus infection of human monocytes by dengue virus-antibody complexes. These results indicate that PBMC from dengue-immune donors contain CD4+ T cells that proliferate and produce IFN gamma after stimulation with dengue antigens, and suggest that the IFN gamma that is produced by these stimulated dengue-specific T cells may contribute to the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome by increasing the number of dengue virus-infected monocytes in the presence of cross-reactive anti-dengue antibodies.

Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue.

It has been reported that the severe complication of dengue virus infection, dengue hemorrhagic fever (DHF) is much more commonly observed during secondary dengue virus infections than primary infections. In order to elucidate the role of T lymphocytes in the pathogenesis of DHF, we attempted to determine whether T lymphocytes are activated in vivo during dengue virus infections, by examining the levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8), interleukin-2 (IL-2) and interferon-gamma (IFN gamma) in the sera of 59 patients with DHF and 41 patients with dengue fever (DF). The levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma were significantly higher in the acute sera of patients with DHF than in the sera of healthy children (P less than 0.001 for all markers). The acute sera of patients with DF contained higher levels of sIL-2R, sCD4, IL-2, and IFN gamma than the sera of healthy children (P less than 0.001 for sIL-2R, IL-2, and IFN gamma; P less than 0.05 for sCD4), but did not have elevated levels of sCD8. The levels of sIL-2R (P less than 0.05), sCD4 (P less than 0.001), and sCD8 (P less than 0.001) were higher in DHF than in DF on days 3-4 after the onset of fever. The levels of IL-2 and IFN gamma in patients with DHF were highest 1 d before defervescence. There were no significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma among grades 1, 2, and 3 of DHF. These results indicate (a) T lymphocytes are activated and produce IL-2 and IFN gamma in vivo during DHF and DF, (b) CD4+ T lymphocytes are activated in DHF and DF, and the level of activation is higher in DHF than in DF, and (c) activation of CD8+ T lymphocytes is evident in DHF, but not in DF.

Dominant recognition by human CD8+ cytotoxic T lymphocytes of dengue virus nonstructural proteins NS3 and NS1.2a.

A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8+ T cell responses to dengue viruses. Memory CD8+CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8+CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue.

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.

Regulatory approach toward vaccines against cancer: the benefit/risk ratio.

Decisions regarding investigational new drug applications for testing of all biologicals to be used in humans, including those designed to prevent or treat cancers, must be considered in terms of benefit/risk ratios. In order for a regulatory organization to make the appropriate decision regarding the testing of new biologicals in humans, it must have an extremely broad dialog within the critical scientific public and the general public, which are represented on committees functioning in the Bureau of Biologics. There must be a generaly consensus regarding the benefit/risk ratio of proposed new biologicals for scientific progress and the effective regulation of biologicals.

Cytokine production by dengue virus antigen-responsive human T lymphocytes in vitro examined using a double immunocytochemical technique.

A number of studies suggest that cytokines may contribute to the pathogenesis of viral infections, including dengue. In this study, we developed a double immunocytochemical method and characterized cytokine-producing cells in the peripheral blood mononuclear cells (PBMC) of dengue virus-immune donors after in vitro stimulation with specific dengue antigens. We found that double immunostaining using immunoalkaline phosphatase (Vector blue) for cytokines [interferon-gamma (IFN-gamma), interleukin (IL) -2, -4, -1alpha, -1beta, and -6, tumor necrosis factor beta (TNF-beta), and TNF-alpha] and immunoperoxidase [diaminobenzidine (DAB)] for cell surface markers (CD3, CD4, CD8, CD20, and CD68) provided the best distinction of double-positive cells from single-positive or -negative cells. The number of IFN-gamma, IL-2, IL-4, and TNF-beta-positive cells increased 2 or 3 days after stimulation with specific dengue antigens. No or very few cytokine-producing cells were detected in the PBMC of non-immune donors stimulated with dengue antigens and the PBMC of immune donors stimulated with a control antigen. The analysis of cell surface markers showed that mainly CD4+ and CD8+ T cells produced these cytokines. The results obtained by immunocytochemistry were consistent with cytokine levels detected in the culture medium assayed by enzyme-linked immunosorbent assay. In conclusion, this double immunocytochemistry technique is suitable for the detection and characterization of cytokine-producing cells in PBMC. Furthermore, the results support the hypothesis that antigen-stimulated CD4+ and CD8+ T cells produce cytokines that may play a role in the pathogenesis of dengue virus infection.

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.

An assay for virus-specific help for B cells.

Spleen cells from mice immunized with vaccinia or influenza viruses were mixed with spleen cells from mice immunized by dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) and transferred into irradiated syngeneic recipient mice which were subsequently restimulated with trinitrophyenylates (TNP) or unmodified viruses. One week later, spleens were removed and assayed for indirect anti-DNP plaque-forming cells (PFC). When spleen cells from both influenza and vaccinia primed mice were restimulated with the haptenated form of the virus used for the initial immunization there was an enhanced PFC response compared to that seen with spleen cells from unprimed mice.