Human immune response to rabies nucleocapsid and glycoprotein antigens.

Antibodies to two components of rabies virus, nucleocapsid (N) and glycoprotein (G), were compared in 11 rabies patients with those in nine recipients of Vero cell rabies vaccine. All rabies vaccinees had antibodies to N and G components by day 10 after the first vaccine injection. A similar but not identical response was observed in three out of 11 rabies patients. Serum antibodies appeared in rabies patients as early as 3 days after onset of the first symptoms of the disease. In these antibody-positive rabies patients, levels of both antibodies, but particularly of anti-N antibody, were lower than in the vaccinated group. Our results suggest that the process of immune recognition and of antibody development in human rabies is more likely to occur early in the pre-clinical phase, and that reactivity to N protein may be crucial for elicitation of neutralizing antibody.     

The cellular immune response to nucleocapsid antigens in hepatitis B virus infection

Conclusions The pathogenesis of HBV-induced hepatocellular injury still remains unsolved despite several years of continuous research effort by a number of investigators. A plausible hypothesis is shown in Fig. 1. The central issue is the role of CTL in causing liver cell death and HBV clearance. Although the HBV nucleoprotein has been suggested to be a major target antigen for CTL [16], it is entirely possible that selected regions of the viral envelope and other nonstructural proteins such as the polymerase may serve as target structure for HLA class I- or class II-restricted CTL recognition, as has been shown in other viral systems. CTL might in turn be negatively modulated by antibody masking of target antigen(s) [22] and by specific intrahepatic suppressor T cells which have been recently demonstrated to be active in chronic HBV infection [4]. In addition, helper T lymphocytes may exert an indirect cytotoxic effect through the release of cytokines such as tumor necrosis factor [5]. This circuit can be potentially amplified by soluble factor(s) secreted by autoreactive cells [5]. Finally, antibody-dependent cell-mediated cytotoxicity (ADCC) may also be a determinant of liver cell necrosis as has been suggested earlier [12].The use of molecular vectors and of synthetic peptides derived from the various viral protein sequences will help us to dissect the immune response to HBV. We anticipate that this strategy will permit us to identify the function, phenotype, HLA restriction and antigenic fine specificity of HBV-specific CTL in HBV infection with the hope that such knowledge may ultimately be translated into more specific and effective therapeutic strategies for eradication of persistent HBV infection and associated liver disease in the future.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Antibody response to outer-membrane antigens of Pseudomonas aeruginosa in human burn wound infection

There is little information about the local and systemic antibody response to surface antigens of bacteria growing in situ in infected lesions in man. In this study, Pseudomonas aeruginosa was obtained directly from the infected wounds of two patients with burns and studied without subculture. Outer-membrane proteins (OMPs) were investigated and compared with those of cells cultivated in the laboratory, with the aim of selecting defined growth conditions to give surface antigens more closely resembling those found in vivo. Several high-mol. wt (77,000-101,000) proteins were expressed in the outer membranes of the bacteria from the patients and could be phenotypically induced by cultivating the same isolate in iron-depleted conditions in vitro. Other major OMPs (D, E, F, G and H) were also observed in cells taken from the lesions. Immunoblotting demonstrated that proteins D and E were recognised by different classes of immunoglobulins in the sera of both patients as was flagellar antigen present in the outer-membrane preparation of the P. aeruginosa from patient 1. Iron-regulated membrane proteins (IRMPs) were similarly detected, but more strongly by IgM from patient 1. Furthermore, a marked antibody response to IRMPs was noted at the site of infection. Bands of a similar intensity were seen after absorption of the sera with lipopolysaccharide (LPS) purified from the infecting strain. This indicated that the response observed was directed against OMPs (including IRMPs) and not against contaminating LPS.     

Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection.

The smooth lipopolysaccharide complex of the outer surface of smooth Brucella abortus cells is believed to be the antigenic component involved in serological tests routinely used for the diagnosis of brucellosis. Sera from cattle vaccinated or infected with B. abortus generally contain antibody directed toward the smooth lipopolysaccharide complex. The brucella organism contains a large number of other antigenically distinct components. The biological significance of some of these antigens has been demonstrated by showing that sera from infected cattle have precipitins to these components. These sera revealed up to seven distinct lines in immunoelectrophoresis with a protein-rich antigen mixture prepared from rough strain B. abortus 45/20, whereas sera from strain 19-vaccinated cattle did not reveal these lines at 4 or more months after vaccination. Monospecific antisera were prepared against six antigens in this mixture, and the purification of two of them by antibody affinity chromatography is described.     

Antibody responses to toxoplasma antigens in mice infected with strains of different virulence.

Swiss Webster mice were infected with either the relatively virulent C56 strain or the relatively avirulent C37 strain of Toxoplasma gondii, and the sequence of their antibody response to surface antigens of the parasite was studied. An immunoglobulin M (IgM) agglutinating antibody was the first serologically detectable antibody and was first detected on day 2 in mice infected with the C37 strain and on day 5 in mice infected with the C56 strain. IgG antibodies were first detected on day 8 for both strains. The major component of the IgG antibodies was IgG2:IgG3 antibodies had lower titers, and no IgG1 antibodies were detected. The IgG antibodies were active in direct parasite agglutination and in the complement-dependent cytotoxicity assay of Sabin and Feldman (Science 108:660-663, 1948). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sera from mice infected with T. gondii detected all major radioiodinated surface proteins of toxoplasma tachyzoites. The earliest time point at which these antigens were detected differed for the two strains. Serum from mice infected with C56 strain immunoprecipitated all cell surface antigens by day 10 of infection, whereas serum from mice infected with the C37 strain did not do so until day 15 of infection.     

The prodigiozan and pyrogenal stimulation of the humoral response to influenza virus antigens in mice

The adjuvant properties of prodigiosan and pyrogenal, lipopolysaccharides of medical importance, were studied. Prodigiosan inoculated to mice together with influenza virus was found, on the one hand, to increase 10-fold the titer of specific antibodies and, on the other, to allow to reduce the antigen dose 50-fold or more. No adjuvant properties of pyrogenal were observed under the experimental conditions.     

Antibody response to influenza vaccination in healthy adults

The aim of this study was to assess antibody response in 184 healthy adults vaccinated with split influenza vaccine (Begrivac, Chiron Behring). Response to hemagglutinin and neuraminidase was assessed before vaccination and after 1 month by hemagglutination inhibition test and neuraminidase inhibition test. After vaccination, statistically significant increases of antibody titers, both for hemagglutinins and for neuraminidases, were observed. The post-vaccination proportion of persons with protective antihemagglutinin antibody titers ranged from 78.4%to 90.8%, while the proportion of persons with at least a fourfold increase of antihemagglutinin antibody titers ranged from 50.5% to 71.2%. All requirements of the Committee for Proprietary Medicinal Products regarding humoral response to inactivated influenza vaccine in healthy adults were fulfilled. Due to a wide range of age of the persons included in this study, the results were also analyzed in two age groups: from 20 to 45 years and from 46 to 56 years. Nevertheless, there were no statistically significant differences in antibody response between these two groups either for hemagglutinin or for neuraminidase.     

Antibody response to polyhistidine-tagged peptide and protein antigens attached to liposomes via lipid-linked nitrilotriacetic acid in mice

Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; K(D) [equilibrium dissociation constant], ～10 μM), trivalent NTA (tris-NTA; K(D), ～1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 10(6) versus 1.4 × 10(6) to 1.6 × 10(6) [P < 0.001]; N-MPR titer, 4.4 × 10(4) versus 5.5 × 10(2) to 7.6 × 10(2) [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines.     

The response to Salmonella typhimurium antigens by two different strains of mice

A technique is described for the detection and enumeration of cells producing antibody to O-somatic antigen specificities 1, 4, 5 and 12 of Salmonella typhimurium. Using this technique, a difference in the immune response has been demonstrated between Swiss White and the BALB/c strain mice, when both these strains are injected with a standard dose of acetone-killed S. typhimurium C5 vaccine. BALB/c mice fail to respond to antigen 5 and their response to the 1, 4 and 12 O-somatic antigens, while reaching the same magnitude, is less prolonged than that in Swiss White mice.     

Antibody response to various single-factor o antigens of salmonella

The relative agglutinin responses to various single O-antigenic (Kauffmann-White) factors were measured after immunization of rabbits with several strains of heat-killed salmonella organisms. As expected, the relative strength of the responses to the various O factors was quite varied and in some cases depended on the presence or absence of other single factors. For example, antibodies to factor 12₂ were formed rapidly and to extremely high levels in rabbits immunized with either Salmonella typhi (O 9,12₁,12₂,12₃) or S. paratyphi B (O 1,4,5,12₁,12₂), whereas factor 12₃ in S. typhi and factor 1 in S. paratyphi B induced only minimal responses. However, rabbits immunized with S. paratyphi A var. durazzo (O 2,12₁,12₃), which lacks factor 12₂, produced high levels of agglutinins to the 12₃ antigenic determinant. In general, most of the agglutinin responses to the various single factors measured were formed in parallel, but there were several exceptions. For instance, the responses to factors 4 and 5 were relatively strong in rabbits receiving three graded doses of S. paratyphi B. However, agglutinins to factor 4 did not appear until after the second injection, and not at all in rabbits given the full amount of antigen in one injection. In contrast, antibodies to factor 4 were formed rapidly in rabbits receiving three graded doses of a strain of S. typhimurium (O 1,4,12) lacking factor 5. Good overall agreement was obtained between agglutination and hemagglutination assays of antibodies, as demonstrated by the responses to the various O factors of S. friedenau. It was concluded that measurement of the antibody responses to the various single-factor O antigens throughout the immunization program was necessary for effective evaluation of the relative significance of these factors in antibody formation against intact bacteria.     

Antibody response to Pseudomonas aeruginosa surface protein antigens in a rat model of chronic lung infection

For an animal model of infection to be useful in immunological studies it is necessary to establish that the surface antigens expressed by bacteria growing in vivo in the experimental infection mimic those expressed by bacteria in the human infection. In this study, chronic infection was induced by inoculating the lungs of rats with agar beads containing mucoid Pseudomonas aeruginosa. P. aeruginosa was obtained from the lungs 14 days after infection and studied without subculture. Several high-mol.-wt proteins were expressed in the outer membranes (OM) of the bacteria from the rat lungs which could be induced by cultivating the same isolate in iron-depleted conditions in vitro. The pattern of iron-regulated membrane proteins (IRMP) was similar to that obtained in an earlier study with another mucoid isolate of P. aeruginosa examined directly, without subculture, from the sputum of a cystic fibrosis patient. Immunoblotting with LPS-absorbed serum from infected rats and also with serum from CF patients showed that IgG in these fluids reacted with the IRMPs and other major OM proteins (OMPs) of P. aeruginosa. Antisera from rats immunised with whole cells of P. aeruginosa grown in iron-depleted media reacted with all the major OMPs of P. aeruginosa, including the IRMPs, confirming their immunogenicity.     

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.     

Cytotoxic activity of normal killers and the lymphocytic proliferative response to specific viral antigens in influenza in mice

Inoculation of CBA mice with pathogenic influenza A/PR8/34 (H1N1) virus and non-pathogenic A/Krasnodar/101/59 (H2N2) virus demonstrated that the pathogenic strain enhanced the synthesis of serum interferon and activated the function of normal killers, but had a relatively low capacity of causing in vitro proliferative response of spleen lymphocytes of intact and in vivo primed animals. In contrast, the nonpathogenic virus had less marked interferon-inducing capacity and that of activating normal killers, but induced a very high proliferative response of lymphocytes in culture.     

Spectrum of antibodies to HCV antigens in patients with different clinical patterns of chronic HCV infection

Correlations between the spectra of antibodies to HCV proteins represented by various antigenic determinants and clinical variants of chronic HCV infection were studied. Synthetic peptides core-16, NS4-20, and NS5-23 simulating the immunodominant regions of the core, NS4 and NS5 proteins and recombinant proteins core-114 and NS4-86 were used as antigens. The results indicate that if the serum of an HCV patients contains no IgG to both antigenic determinants of NS4 or to NS5 in combination with any core antigenic determinant, a clinical and biochemical remission is highly probable. Chronic hepatitis C is characterized by the presence of IgG in high titers to both antigenic determinants of NS4 protein, particularly in combination with anti-NS5 IgG in low titers or none at all, or high titers of anti-core-16 IgG in combination with high titers of anti-NS4-20 IgG.     

Lymphocyte blastogenic responses to influenza virus antigens after influenza infection and vaccination in humans.

Virus-specific in vitro cell-mediated immune responses were investigated in 20 normal volunteers who were challenged with liver influenza A/VIC/3/75 (H3N2) virus and in 13 volunteers who were vaccinated with inactivated vaccine containing A/VIC and A/NJ/8/76 (HswN1) antigens. Lymphocyte cultures were established from peripheral blood samples obtained prior to and at various times after infection or vaccination. Blastogenesis was determined by [3H]thymidine incorporation after stimulation of cultures with purified, inactivated, whole influenza viruses. Six days after infection, significantly elevated levels of blastogenesis were observed after in vitro stimulation with A viruses of hemagglutinin and neuraminidase subtypes that were the same as (H3N2) or antigenically distinct from (Heq1Neq1 or HswN1) those of the challenge virus, although maximum stimulation was noted with virus of the same hemagglutinin subtype (H3) as the challenge virus. Similar although more prolonged blastogenic responses were noted in lymphocyte cultures from vaccinees who had serum antibody rises after vaccination. The kinetics of these responses suggest that cell-mediated immunity may play a role in early events after infection and vaccination with influenza virus.     

The immune response in NZB mice of different ages to thymus-dependent and thymus-independent phosphorylcholine antigens.

This study was designed to examine aberrations of immune responses in autoimmune NZB strain mice during ageing, at the level of individual B cell clones. The response to phosphorylcholine (PC) was chosen because murine responses to PC are restricted to a few B cell clones and can be characterized with idiotypic markers. Responses to both thymus-dependent (TD) and thymus-independently (TI) PC-containing antigens were measured in mice ranging from 1 to 62 weeks of age. We found that: (1) TD responses to phosphorylcholine keyhole limpet haemocyanin (PC-KLH) decreased markedly (about 17-fold) between 28 and 62 weeks of age. TI responses to the R36a strain pneumococcus decreased only slightly during the same period. (2) The PFC responses to both antigens became markedly prolonged in mice older than 24 weeks. (3) The NZB response to either antigen is essentially monoclonal, as measured by inhibition of PFC with specific anti-idiotype serum and PC hapten. No age-related alteration in avidity or idiotype expression was observed. Our results demonstrate that no aberrant PC-reactive B cell clones appear in old NZB, and lend support to the notion that the abnormalities observed are due to defective regulatory mechanisms.