IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

Protective Cellular Immunity Against Influenza Virus Induced by Plasmid Inoculation of Newborn Mice

Neonate organisms display an intrinsic disability to mount effective immune responses to infectious agents or conventional vaccines. Whereas low. doses of antigens trigger a suboptimal response, higher doses are frequently associated with tolerance induction. We investigated the ability of a plasmid-expressing nucleoprotein of influenza virus to prime a specific cellular immune response when administered to newborn mice. We found that persistent exposure to antigen following plasmid inoculation of neonates leads to a vigorous priming of specific CTLs rather than tolerance induction. The CTLs were cross-reactive against multiple strains of type A influenza viruses and produced IFNγ but no IL-4. The immunity triggered by plasmid inoculation of neonates was protective in terms of pulmonary virus clearance as well as survival rate following lethal challenge with influenza virus. Whereas the persistence of the plasmid at the site of injection was readily demonstrable in adult mice at 3 months after inoculation, mice immunized as newborns displayed no plasmid at 3 months and very little at 1 month after injection. Thus, DNA-based immunization of neonates may prove an effective and safe vaccination strategy for induction of cellular immunity against microbes that cause serious infectious diseases in the early period of life.     

Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities ( approximately 108 M(-1)) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications.     

Influence of active immunization of rats with conjugated serotonin-protein antigen on the content of biogenic amines in the brain and on behavioral responses

The influence of induction of antibodies to the neuromediator serotonin in the case of active immunization of animals with a conjugate serotonin-protein antigen on the distribution of biogenic amines in the brain and on behavioral responses was investigated in experiments on rats. It was shown that active immunization of rats with a serotonin-protein conjugate leads to a decrease in serotonin, its metabolite 5-hydroxyindole-3-acetic acid, as well as dopamine in certain brain structures. Against a background of induction of antibodies to serotonin, the horizontal motor activity of the animals is decreased.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 74, No. 10, pp. 1367–1372, October, 1988.     

Anti-idiotypic IgM antibodies to anti-HLA class I antibodies in habitual abortion.

In order to investigate the role of the idiotypic network in miscarriages, sera from 28 habitually aborting women undergoing paternal leukocyte immunization were studied for the presence of HLA antibodies and related anti-idiotypes. Sixty-eight percent of sera from preimmunized patients which did not contain anti-lymphocyte antibodies inhibited the activity of antibodies to the HLA class I antigens expressed by the spouse. This inhibitory activity could be assigned to IgM antibodies, which cross-inhibit antibodies of similar specificity. This suggests that they are anti-idiotypes for the binding site of HLA antibodies. Immune sera of successfully treated patients exhibited both cytotoxic IgG anti-HLA antibodies and inhibitory IgM anti-idiotypic antibodies. A possible role for an intact idiotypic network in maintaining pregnancy is suggested.     

Priming B cell-mediated anti-HIV envelope responses by vaccination allows for the long-term control of infection in macaques exposed to a R5-tropic SHIV

The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIV(SF162P4). Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection.     

Identification of epididymis-specific antigens using neonatal tolerization

PROBLEM: Conventional immunization using whole sperm containing multiple antigens as the immunogen followed by hybridoma technology usually gives antibodies to antigens invariably of testicular origin, probably because of the strong immunogenic nature of these antigens. Therefore, an alternate approach of neonatal tolerization or subtractive immunization has been utilized to raise antibodies specific to epididymis by suppressing immune response to testicular antigens. METHOD OF STUDY: Neonatal mice were tolerized with testicular sperm proteins on days 0 and 5. These animals were then immunized with epididymal sperm proteins on day 21, followed by two boosters at biweekly intervals. Sera from these mice were used to localize epididymis-specific antigens. RESULTS: Sera from mice that were tolerized to testicular sperm proteins and later immunized with epididymal sperm proteins reacted only with epididymal proteins. CONCLUSION: The results of this study demonstrate that neonatal tolerization with testicular sperm proteins, followed by immunization with epididymal sperm proteins, enhances the production of antibodies to proteins exclusively of epididymal origin.     

Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter

In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders.     

Immune response of adults to sequential influenza vaccination

Annual immunization against influenza is recommended for numerous individuals, but the antibody response to sequential vaccination has not been well characterized. Levels of hemagglutination-inhibition antibody were measured in adults given either two or three doses of trivalent influenza vaccine at six-month intervals. A significant rise in the number of individuals with antibody titers of greater than or equal to 40 was seen for all three antigens only after initial vaccination. Repeated vaccination was necessary to maintain adequate antibody levels only to the A/Brazil (H1N1) antigen; it did not significantly affect the proportions of individuals with protective levels of antibody to either the A/Bangkok (H3N2) or the B/Singapore 222/79 antigens. These findings do not support the current recommendation for annual immunization when the vaccine formulation has not changed.     

Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children

Because 21 immunized children (13%) among the 162 confirmed Japanese encephalitis (JE) cases during 1986–1991 occurred in Taiwan, we collected 320 serum samples from Taiwan children aged 15–31 and 27–44 months immediately before the 1st dose (n = 41) and 1–3 months after the 2nd dose (n = 78, 27 pairs), and immediately before (n = 58) and 1–3 months after the 3rd dose (n = 143,44 pairs) to determine neutralization antibody (Nt Ab) against the Nakayama (N) and Beijing-1 (B) strains and two Taiwan wild type JE viruses (JEV): CC-27 and CH-1392. Our Nt results showed that (1) B vaccine stimulated a better homologous Ab response than N vaccine for Nt Ab seropositivity rate (NASR), produced a higher level of Nt titer after the primary immunization [2 doses = 100% vs. 91%, geometric mean titer (GMT) = 115 vs. 22], had a greater booster effect (3 doses: 100% vs. 95%; GMT = 320 vs. 33), and showed a better capability to neutralize two local Taiwan JEV strains, particularly only after 3 doses (ave. NASR for B vs. N = 90% vs. 10%; and GMT for B vs. N = 154 vs. 1); (2) the two wild type JEV strains had different plaque morphology and antigenic variation and the CC-27 strain was not neutralized as well as the CH-1392 strain after 3 doses of vaccine (BBB or NNN or NNB); and (3) 30% of the children had lost JEV Nt Ab one year after the 2nd dose of N vaccine and natural infection with JE virus did occur among those children after immunization. In conclusion, (1) three doses of mouse-brain vaccine are the minimum requirement to protect children against the local Taiwan JEV; (2) the best strain for a JE vaccine depends on level of Nt Ab it induced, the molecular epidemiology and antigenic variation of the JEV in each local area; and (3) future vaccine must produce better B- and T-cell memory. © 1994 Wiley-Liss, Inc.