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Thirty-one unprimed children and young adults received an influenza A/USSR/77 vaccine containing 43 microgram of hemagglutinin. Their HAI antibody response was compared to that in 92 age-matched individuals from the 1978 national influenza immunization trial who received 10 and 4 microgram HA vaccines. A dose-related antibody response was observed after the first vaccine doses in the 7- to 12- and 13- to 25-year-old groups. An HAI titer greater than or equal to 40 was present in 81% and 93% of 43 microgram HA recipients, in 38% and 43% of 10 microgram HA recipients, and in 24% and 12% of 4 micrograms HA recipients respectively. The antibody response to the 43 microgram HA dose was significantly higher than was the response to the 10 and 4 microgram HA doses. The local and systemic side effects were not significantly different among the three vaccine groups and the placebo group. Thus, a high dose of influenza A/USSR/77 split-product vaccine given to unprimed children and young adults stimulated presumably protective levels of antibody and was free of a significant incidence of side effects.

Purified concentrates of influenza A/USSR/90/77(H1N1)-like, A/Texas/1/77 (H3N2)-like, and B/Hong Kong/5/72-like viruses used for preparation of investigational and licensed vaccines in 1978 to 1979 were tested for their content of neuraminidase enzyme activity. Concentrates of H1N1 virus used to prepare vaccines for clinical investigations performed in the spring of 1978 had neuraminidase activity at that time which decreased during storage to almost undetectable levels (three lots) or by 50% (one lot) by the winter of 1978. Several other lots of concentrates prepared with H1N1 virus and used for vaccine formulation had no detectable neuraminidase enzyme activity when tested in the winter of 1978, at a time when they would be administered in vaccines. The range of specific activity for different lots of concentrates was about 40-fold for A/Texas/1/77, B/Hong Kong/5/72, and A/USSR/90/77 neuraminidases. Immunogenicity of investigational vaccines prepared with tested concentrates and administered between April and July 1978 was measured in volunteers aged 13 to >50 years. Frequency of neuraminidase antibody rises to two doses of H1N1-containing vaccine was 10% in unprimed subjects aged

Summary Cell-mediated immunity is important in host control of CMV infection. A chromium release microcytotoxicity assay was used to evaluate the role of cytotoxic T lymphocytes (CTL) in murine CMV infection. Within a few days after intranasal inoculation virus was detected in cultures of buffy-coat, spleens, anterior cervical lymph nodes and salivary glands. CTL were first detected on day 5 post-infection in spleen and peripheral blood, and on day 6 in anterior cervical nodes. The course of the CTL response approximated to that of virus titres during the acute phase of infection in the spleen and blood. The findings indicate that CTL are distributed to infected tissues and appear to be important during the acute, viraemic phase of infection.

SummaryReplication of herpes simplex virus in lymphocyte cultures stimulated by PWM, Con A, and ALG is compared with the replication of this virus in PHA-stimulated lymphocytes. The resulting virus titers are similar with all the mitogens tested and appear to be dependent on the amount of DNA synthesized by the cells rather than on blastogenesis.

Recombinant Japanese encephalitis (JE) vaccine candidates based on a highly attenuated vaccinia virus (NYVAC-JEV) and a canarypox virus (ALVAC-JEV) were evaluated for their ability to induce specific antibodies and cytotoxic T lymphocytes (CTLs) in mice. Six- to eight-week-old male Balb/c mice that received one or two intraperitoneal inoculations with these JE vaccine candidates at a dose of 1 × 10 7 PFU per mouse produced neutralizing antibody and antibodies to the envelope (E) and nonstructural 1 (NS1) proteins as determined by radioimmunoprecipitation. Immunization with either of these vaccine candidates also induced JE virus-specific T lymphocytes that proliferated in response to stimulation with infectious virus and/or noninfectious viral antigens. Mice maintained detectable levels of neutralizing antibody and JE virus-specific memory T cells for at least 6 months after immunization with NYVAC-JEV and for 4 months after immunization with ALVAC-JEV. Cells induced to proliferate after stimulation with live virus contained specific CD8 + CTLs that lysed primary Balb/c mouse kidney cells infected with JE virus and P815 mastocytoma cells infected with a recombinant vaccinia virus expressing the premembrane (prM), E, and NS1 proteins. These CTLs also lysed P815 cells infected with vaccinia recombinants expressing prM and E, and those expressing E and NS1, but did not lyse P815 cells infected with a recombinant virus expressing only NS1, indicating that the CTLs mainly recognized E, but did not recognize NS1. These results demonstrate that both recombinant JE vaccines, NYVAC-JEV and ALVAC-JEV, induce JE virus-specific antibody and CTLs in mice.

There is a need to improve the ability of subunit vaccines to induce CD8+ CTL responses in humans, especially for vaccines used to prevent illness by organisms that undergo antigenic variation at their major neutralizing antibody sites, e.g., influenza A viruses and human immunodeficiency virus. Murine models have demonstrated the protective role of cross-reactive CTL against influenza A virus antigenic drift. We tested the ability of an adjuvanted carrier (Iscomatrix) to help human antigen-presenting cells present formalin-killed influenza vaccine to human CD8+ CTL clones in vitro and in vaccinated humans. The results of a randomized, double-blind, controlled clinical study demonstrate that a single dose of a vaccine formulated into Iscom particles increased influenza A virus-specific CTL memory in 50–60% of recipients, compared to 5% of the recipients of the standard influenza vaccine.

We examined nine dengue virus-specific human CD4+ CD8− cytotoxic T lymphocyte (CTL) clones for protein recognition, using recombinant vaccinia viruses which contain genes coding for dengue virus proteins. These clones were established from peripheral blood mononuclear cells of a donor previously immunized with a live-attenuated experimental dengue-1 vaccine. Of nine CD4+ T cell clones, seven were dengue-1-specific and two were dengue-1–dengue-3 cross-reactive. Four dengue-1-specific clones and one dengue-1–dengue-3 cross-reactive clone recognized epitopes within the NS1 or NS2a proteins. Analysis of human leukocyte antigen (HLA) restriction revealed that three dengue-1-specific clones are HLA-DR1-restricted and one dengue-1–dengue-3 cross-reactive clone is HLA-DPw3-restricted. These results indicate that NS1 and NS2a proteins as well as C, E, and NS3 proteins reported earlier contain one or more epitopes recognized by dengue virus-specific human CD4+ T lymphocytes.

THE immune response to cytomegalovirus (CMV) infection is poorly understood, but it is believed that the cellular immune system has a critical role. It is a major cause of morbidity and mortality in homograft recipients1,2, leukaemic individuals3,4 and others receiving immunosuppressive drug therapy5. In these patients, high levels of serum antibody often fail to eradicate chronic infection6. Infection of mice with murine cytomegalovirus (MCMV)7 has provided additional evidence of the importance of T-lymphocyte function in the immune response to CMV: nude mice8, and mice treated with anti-thymocyte serum9 or undergoing graft-versus-host reaction10 all succumb to much lower infecting doses of virus than do normal mice. It has been shown that purified human CMV can induce proliferation of lymphocytes from immune individuals11, but T-cell specific responses to CMV infection have not yet been demonstrated. Cytotoxic T-lymphocyte responses have been described for several viral infections12–17. We report here a virus-specific cytotoxic T-lymphocyte (CTL) response in MCMV infection.

A double-blind randomized study with bivalent influenza virus vaccines was conducted to compare the local and systemic reactions and immunogenicity of a whole-virus vaccine and a split-product vaccine in children. Fevers of greater than 100 F were more frequent after vaccination with whole-virus than split-product vaccine especially in children one to four years old (69% vs 22%; P less than 0.01). Fevers of greater than or equal to 103 F did not occur in children who previously had been given influenza virus vaccine, even in the absence of preexisting homologous serum antibody. The immune response to the A/Port Chalmers/1/73 antigen in the vaccine was similar after administration of either whole-virus or split-product vaccine. However, split-product vaccine induced significantly less hemagglutination-inhibiting antibody to B/Hong Kong/5/72 virus in children younger than 10 years who had not been previously immunized; only 43% developed detectable antibody vs. 100% of those vaccinated with whole virus vaccine (P less than 0.01). These studies indicate that (1) in young children whole-virus vaccine causes fever more frequently than split-product vaccine; (2) young children previously vaccinated with influenza virus vaccine are unlikely to experience fever subsequent to immunization with a related antigen; and (3) split-product vaccine induces less antibody to B/Hong Kong/5/72 virus than whole-virus vaccine in immunologically unprimed young children.

Rimantadine HCl was assessed for its effect on influenza A virus titer in lungs of infected BALB/c mice. Rimantadine administered orally via drinking water, with and without an intraperitoneal prophylactic loading dose, was compared to intraperitoneal administration. Mice were infected with a non-lethal dose of influenza A/Port Chalmers/H3N2 virus and the pulmonary virus titers were determined at intervals over a 21 day period. Prophylactic treatment with rimantadine followed by oral administration resulted in up to a 4 log10 reduction in pulmonary virus titer. The oral doses given to the mice were comparable on a mg/kg/day basis to those recommended for treatment of human infections. Reductions in pulmonary virus titers also occurred after intraperitoneal rimantadine treatment which included a prophylactic dose, but the reductions in pulmonary virus titers were less striking and not consistent over the course of infection. There were no significant reductions in pulmonary virus titers by either route if treatment was started 8 h after exposure to virus.

Latent murine cytomegalovirus (MCMV) infection of BALB/c mouse spleens was studied using several methods including an explant tissue culture technique, co-cultivation on allogeneic and syngeneic cell cultures and nucleic acid hybridization. BALB/c mice experience latent infection which persists for at least 6 months and involves only a small fraction of spleen cells. The explant culture technique proved to be much more sensitive than other methods for detecting latent infection of lymphoid tissues.

We recently reported that influenza infection of humans induced interferon and augmented levels of natural killer cell activity. 1 We also detected a virus-specific HLA restricted cytotoxic T lymphocyte response in humans given live attenuated or inactivated influenza vaccines. 2 In addition to the virus-specific HLA restricted lysis by the volunteers' lymphocytes of 51 Cr labelled target cells, we noted a lower degree of non-HLA restricted lysis which was not virus specific. 2 Since it had been reported that influenza infection in vivo 3 and in vitro 4 could induce interferon, we speculated that the nonspecific cytotoxicity was due to augmented natural killer cell activity by the cultured lymphocytes as a result of interferon production in the culture. The titers of interferon produced by the lymphocytes stimulated by influenza-infected stimulator cells were >10,000 U/ml. The interferon was pH 2 labile and was not neutralized by antisera to alpha or beta interferon. 5 It did not bind to a monoclonal antibody to alpha interferon, and was neutralized by antiserum to gamma interferon. The influenza antigen responsible for stimulating this response is a crossreactive determinant present on all human and non-human influenza viruses tested, and some preparations of purified hemagglutinin do not induce this immune interferon.

This report summarizes the clinical trials of the A/USSR/77 (H1N1) influenza vaccines performed in 1978. A total of 2,091 subjects participated in these trials. The results of these clinical trials indicated that two doses of H1N1 viral antigen were necessary to produce serum titers of hemagglutinin-inhibiting (HAI) antibody of greater than 1:40 in 80% or more of the test subjects younger than 25 years of age, who were unlikely to have experienced natural infection during the earlier period of prevalence of H1N1 virus (1947-1957). Only one dose of the A/Texas/77 (H3N2) or B/Hong Kong/72 antigen was necessary to stimulate equivalent titers of HAI antibody in serum. Thus, previous natural exposure to H1N1 viruses primed individuals 26 years of age or older to respond to H1N1 antigens. No major differences in antigenicity were noted between whole-virus and split-virus vaccines. No differences in reaction indexes measuring systemic reactions were noted when vaccine types were compared. Only one vaccine was associated with a reaction index appreciably higher than that of placebo. The relatively uniform antibody responses observed were attributed to the newer methods of vaccine standardization introduced after the clinical trials in 1976. No cases of vaccine-related neurological problems, including Guillain-Barre syndrome, were found during these trials. Vaccines containing 7-21 micrograms of each viral antigen were antigenic and were well tolerated.

The serologic responses after two sequential nonreactive doses of either chemically disrupted or whole-virus influenza A/New Jersey/76 virus vaccine were evaluated in 112 normal young adults. In general, levels of hemagglutination-inhibiting (HAI) antibody were low after the first dose of vaccine and increased significantly (P < 0.05) in response to a second dose. Whereas one dose of the preparation from Merck Sharp and Dohme (West Point, Pa.) effectively vaccinated this population, two doses of the vaccines prepared by Parke, Davis and Company (Detroit, Mich.) and MerrellNational Laboratories (Cincinnati, Ohio) were required to produce a similar serologic response. The preparation from Wyeth Laboratories (Philadelphia, Pa.) produced low levels of HAI antibodies even after two doses. These different serologic responses correlated with the viral hemagglutinin content of each vaccine. Early clinical trials with the influenza A/New Jersey/76 virus vaccines showed that the serologic response was strongly influenced by the age of the vaccinees and the presumed previous exposure to related influenza A viruses [1]. Whereas a good antibody response was obtained with a single, nonreactive dose of vaccine in adults over the age of 24 years, younger persons showed a poor serologic response to the same dose of vaccine. In addition, the chemically disrupted (split-virus) vaccines were even less immunogenic than the whole-virus preparations in the younger vaccinees. This study was therefore designed to evaluate whether the sequential administration of two nonreactive doses of either disrupted or whole-virus A/NJ/76 vaccine would produce an acceptable serologic response in this relatively nonresponsive population.

Sensitized murine spleen cells decrease the spread of influenza infection in cell cultures derived from human and syngeneic murine tissues. This control of infection is immune-specific and can be induced by immunization with identical hemagglutinin or neuraminidase antigens. The antiviral effect is evident in the absence of added immune serum and can be blocked by adsorption of sensitized spleen cells with antiserum to murine IgG. The antiviral effect persists despite separation by agar of the sensitized spleen cells from the virus-infected monolayer. Spleen cells that do not adhere to glass are quantitatively more capable of limiting the spread of influenza virus in cell culture than are glass-adhering cells. These results suggest that mice immunized with influenza virus have sensitized spleen cells that elaborate antibody to influenza virus, which can act locally to control the spread of infection.

The purpose of the research program is to define immune responses of humans to Human Immunodeficiency Virus (HIV) infection and the cause of the Acquired Immune Deficiency Syndrome (AIDS). Immunodeficiency caused by HIV infections may be due to immune mediated lysis (by antibody and/or HIV specific cytotoxic T lymphocytes CTL) of infected macrophages and T4 lymphocytes. The definitions of these immune responses to HIV virus are needed for rational approaches for therapy and prevention. Since the project began in October, 1986, we have developed an integrated program of research projects to develop experimental systems required for defining the immune responses of humans to HIV. Keywords: AIDS; HIV-1; Retrovirus; Lentivirus; Immunology.

The antibody and HLA-restricted virus-specific cytotoxic T lymphocyte (CTL) responses to influenza vaccine of 36 volunteers were analysed. Three vaccines were used: a live attenuated, and two types of inactivated, a whole virus and a purified surface antigen vaccine. Antibody to haemagglutinin (HA) was assayed using plaque neutralization, single-radial-haemolysis and haemagglutination-inhibition (HI) techniques. Antibodies to nucleoprotein and matrix antigens were also measured. Most of the volunteers had antibody responses to the HA in the inactivated vaccines which were detected by all three techniques. Nine of the twelve recipients of the live virus vaccine did not have an antibody response detected by the HI test, but four of these did have antibody responses when the plaque neutralization test was used. Single-radial-haemolysis was more sensitive than the HI test for detecting low levels of antibody, but the plaque neutralization test was the most sensitive for detecting low levels of antibody. Most volunteers had a rise in their HLA-restricted influenza-specific memory CTL response, but three recipients of live vaccine who did not have an antibody response by any technique also did not have an increase in their cytotoxic T cell activity. Three volunteers, two of whom had received live vaccine, had a positive CTL response without antibody response.

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.

We have studied the kinetics and specificity of the cytotoxic T lymphocyte (CTL) response to influenza A/PR/8 (H1N1) virus pulmonary infection in the mouse detected using spleen cells from infected mice which were stimulated in bulk and limiting dilution cultures. A hybrid protein designated D-peptide, which contains the terminal 157 amino acids of the HA2 subunit of A/PR/8 virus, was used to stimulate influenza virus subtype-specific secondary CTL in vitro. Infection induced two specificities of precursor CTL, cross-reactive and subtype-specific. The kinetics of the subtype-specific CTL response detected by the D-peptide were similar to the cross-reactive CTL response detected by stimulation with live virus. The majority of the precursor CTL (CTL-p) are able to lyse virus-infected target cells in a cross-reactive fashion. The number of memory subtype-specific and cross-reactive CTL increased by approximately 2.5 logs10 during the first 3 weeks after infection.

Four hundred twenty-six volunteers aged 16 to 83 years were given experimental doses of inactivated monovalent [A/USSR/77 (H1N1)] and trivalent [A/USSR/77 (H1N1), A/Texas/77 (H3N2), B/Hong Kong/72] influenza vaccines as part of a double-blinded, placebo-controlled, national collaborative study. Local reactions at inoculation sites occurred in less than 6.6% of volunteers. Systemic reactions, generally consisting of low-grade fever, myalgia, or headache, lasting less than 24 hr, did not occur more frequently in vaccinees than in placebo recipients. Vaccines containing lower doses of the A/Texas/77 (H3N2) hemagglutinin (8-10 micrograms) and B/Hong Kong/72 hemagglutinin (6-9 micrograms) produced hemagglutinin-inhibiting (HAI) antibody responses equivalent to those produced by higher doses (15-28 and 19-28 micrograms, respectively) in all age groups. Single injections of vaccines containing lower doses of A/USSR/77 (H1N1) hemagglutinin (4-7 micrograms) induced titers of HAI antibody of greater than 1:40 in 93% of volunteers greater than or equal to 26 years of age but in only 58% of volunteers less than 26 years of age. In this latter group, two injections containing higher doses (10-19 micrograms) induced better antibody responses. Vaccines containing approximately 6-15 micrograms of hemagglutinin of each antigen can be expected to be well tolerated and to induce good HAI antibody responses in a one- or two-dose regimen, depending on the age group.

R E C E N T L Y M O D I F I E D R E C O M M E N D A T I O N S 1' ~ f o r the use of live attenuated measles vaccines call for routine immunization of children at 15 months of age or older. During epidemics, infants may be immunized at 6 months of age or older, but must be reimmunized at 15 months. A change in the age recommended for administration, from 12 to 15 months was originally suggested by the retrospective observations of Yeager and associates. 3 This report presents the results of two studies designed to study prospectively the relationship between age at immunization and response to measles vaccine. The first study, done during 1974 and 1975, measured seroconversion rates in 6to 12-month-old children. The second study measured seroconversion following measles vaccine given as measles-mumps-rubella vaccine at 12 months of age simultaneously with trivalent oral polio vaccine.

Definition of the T-lymphocyte responses to dengue viruses should aid in the development of safe and effective vaccines and help to explain the pathophysiology of dengue hemorrhagic fever and dengue shock syndrome. In this study, we demonstrated that dengue virus-specific T lymphocytes were detected in spleen cells from dengue virus-immune mice using an in vitro proliferation assay. Following immunization with a single dose of infectious dengue virus, murine lymphocytes showed increased proliferation when incubated in the presence of viral antigens of the same serotype but not in the presence of control antigens. Depletion experiments with antibody and complement showed that the population of responding cells expressed the Thy1+ L3T4+ Lyt2- phenotype. This indicates that the predominant proliferating cells are T lymphocytes of the helper-inducer phenotype. Dengue virus-specific memory lymphocyte responses were detectable for at least 22 weeks after immunization. The response to primary infection was primarily serotype specific, with some serotype cross-reactivity present at a low level. We demonstrated that lymphocytes from mice immunized with dengue 4 virus proliferate in response to a combination of dengue 4 virus C, pre-M, E, NS1, and NS2a proteins expressed in Sf9 cells with a recombinant baculovirus, and, to a lesser extent, to the dengue 4 virus E protein alone.

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexes after rIFN-gamma treatment. Pretreatment of U937 cells with rIFN-gamma resulted in a significant increase in the number of dengue virus-infected cells and in the yield of infectious virus. rIFN-gamma did not augment dengue virus infection when cells were infected with virus in the absence of anti-dengue antibodies. Gamma interferon (IFN-gamma) produced by peripheral blood lymphocytes from dengue-immune donors after in vitro stimulation with dengue antigens also augmented dengue virus infection of U937 cells. IFN-gamma did not augment dengue virus infections when cells were infected with virus in the presence of F(ab')2 prepared from anti-dengue immunoglobulin G. Human immunoglobulin inhibited IFN-gamma-induced augmentation. IFN-gamma increased the number of Fc gamma receptors on U937 cells. The increase in the percentage of dengue antigen-positive cells correlated with the increase in the number of Fc gamma receptors after rIFN-gamma treatment. These results indicate that IFN-gamma-induced augmentation of dengue virus infection is Fc gamma receptor mediated. Based on these results we conclude that IFN-gamma increases the number of Fc gamma receptors and that this leads to an augmented uptake of dengue virus in the form of dengue virus-antibody complexes, which results in augmented dengue virus infection.

Recently, Johnson et al. [1] reported the induction of hydrocephalus in suckling hamsters by intracerebral (IC) inoculation with mumps virus obtained from the cerebrospinal fluid of patients with mumps. The present report confirms and extends Johnson's findings. Here, the effects of IC inoculation of a low-passaged virulent mumps strain, and a higher-passaged strain which is known to be altered as determined by tissue culture marker studies and by vaccination in humans [2], were studied and compared.

Sensitized mouse spleen cells decrease the spread of herpes simplex virus infection in cell culture lines derived from human and murine tissues. These washed, sensitized cells act alone and additively in combination with antibody to diminish the ability of single virus-infected cells to spread infection to contiguous cells. This control of infection is not species specific, unlike interferon, and appears to be distinct from the effect of antibody. Lymphotoxin was not detected in this lymphocyte-mediated response. This control of herpes simplex virus infection in vitro by sensitized lymphoid cells is immunologically specific; spleen cells from donor animals immunized with a heterotypic virus do not cause herpesvirus plaque size reduction. The ratio of spleen cells from immunized animals to target monolayer cells needed to produce this effect is > 4:1. Plaque size reduction of herpes simplex virus by spleen cells requires intact, immune, non-glass-adhering lymphoid cells.

The role of cell-mediated protection against infection with herpes simplex virus was studied by adoptive immunity. Sensitized spleen cells, obtained from syngeneic mice immunized with herpes simplex virus, protected recipient mice against lethal challenge with herpes simplex virus. Survival was significantly greater in challenged mice that had received washed spleen cells from donor animals immunized with herpes simplex than from control donors. This increase in survival after challenge was associated with a significant decrease in titers of virus in the brains of mice that had received specifically sensitized cells. The decrease in viral titers was evident during the period after challenge when titers of virus peak and most deaths occur. There was no obvious association between increased survival and the levels of antibody and interferon in serum of the challenged animals, but these serum factors still must be considered as possible mediators of the protection conferred by sensitized spleen cells.

SummaryThe mumps plaque neutralization assay procedure was found to be a sensitive indicator of low levels of mumps antibodies. Undiluted serum can be studied for the presence of mumps antibodies and there is no problem of nonspecific inhibition. The levels of antibody 1 year after natural mumps are more stable by the PN method than by HAI or CF tests. This assay procedure is valuable in studying sera where low levels of antibody may be present, as after remote natural disease, or vaccination with attenuated mumps strains.

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.

The induction and maintenance of T cell memory is incompletely understood, especially in humans. We have studied the T cell response and the generation of memory during acute infection by the Puumala virus (PUUV), a hantavirus endemic to Europe. It causes a self-limiting infection with no viral persistence, manifesting as hemorrhagic fever with renal syndrome. HLA tetramer staining of PBMC showed that the CD8+ T cell response peaked at the onset of the clinical disease and decreased within the next 3 wk. Expression of activation markers on the tetramer-positive T cells was also highest during the acute phase, suggesting that the peak population consisted largely of effector cells. Despite the presence of tetramer-positive T cells expressing cytoplasmic IFN-γ, PUUV-specific cells producing IFN-γ in vitro were rare during the acute phase. Their frequency, as well as the expression of IL-7Rα mRNA and surface protein, increased during a follow-up period of 6 wk and probably reflected the induction of memory T cells. Simultaneously with the PUUV-specific response, we also noted in seven of nine patients an increase in EBV-specific T cells and the transient presence of EBV DNA in three patients, indicative of viral reactivation. Our results show that in a natural human infection CD8+ memory T cells are rare during the peak response, gradually emerging during the first weeks of convalescence. They also suggest that the boosting of unrelated memory T cells may be a common occurrence in human viral infections, which may have significant implications for the homeostasis of the memory T cell compartment.

Although the swine-influenza-immunization campaign has been suspended for all but high-risk groups, it was attended by other problems besides the well publicized but very rare apparent association with Guillain-Barre syndrome. One of these other problems relates to the difference between split-product and whole-virus types of vaccines, a difference not generally appreciated by physicians. The problem is worth discussing because the basic principles apply to the manufacture of influenza vaccines other than the current swine-virus vaccine. Different manufacturing processes have been developed in the hope of minimizing reactivity (systemic type, fever, malaise, headache and myalgia; local type, arm pain, redness and . . .

Summary Mumps virus was isolated from the saliva 7 days prior to the onset of parotitis in a boy with natural mumps infection. This report of the isolation of mumps virus during the incubation period of natural infection agrees with the finding of salivary shedding of virus following experimental infection with a low egg passage, virulent strain of mumps virus. 5