Role of gamma interferon in natural clearance of Bordetella pertussis infection.

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.

Plasmodium yoelii: 17-kDa hepatic and erythrocytic stage protein is the target of an inhibitory monoclonal antibody.

Infected hepatocytes are important targets for malaria vaccines. To identify Plasmodium yoelii proteins expressed in infected hepatocytes, we immunized BALB/c ByJ mice with P. yoelii liver stage schizonts and produced a panel of monoclonal antibodies (Mabs). An IgG1 Mab, navy yoelii liver stage 3 (NYLS3), had the strongest reactivity against liver stage parasites and was selected for further characterization. The Mab does not recognize P. yoelii sporozoites, but recognizes liver stage parasites within 6 hr of invasion of mouse hepatocytes and throughout the hepatic and asexual erythrocytic stages of the parasite life cycle as determined by the immunofluorescent antibody test. This Mab is species-specific, and it reacts with liver stages of P. yoelii but does not react with liver stages of other Plasmodium species. The protein recognized by this Mab is present on the parasitophorous vacuole membrane of infected hepatocytes and erythrocytes as demonstrated by immunoelectron microscopy and has a relative molecular weight of 17 kDa as demonstrated by immunoblot of an extract of infected erythrocytes. It is therefore designated P. yoelii hepatic and erythrocytic stage protein, 17 kDa or PyHEP17. When added to primary cultures of mouse hepatocytes 24 hr after inoculation with P. yoelii sporozoites, when all sporozoites have invaded hepatocytes, NYLS3 eliminates up to 98% of liver-stage parasites. Intravenous injection of NYLS3 into mice delays the onset and reduces the density of blood-stage parasitemia after sporozoite or blood-stage challenge. The P. falciparum and P. vivax homologs of PyHEP17 may therefore be important targets for vaccines designed to attack the hepatic and erythrocytic stages of the parasite life cycle.

Analysis of protective and nonprotective monoclonal antibodies specific for Bordetella pertussis lipooligosaccharide.

In this study, it has been determined that immunoglobulin G1 (IgG1) and IgG3 monoclonal antibodies directed to the lipooligosaccharide A of Bordetella pertussis were able to protect mice from fatal aerosol infection. No correlation was found between the bactericidal activity in vitro in the presence of complement and the protection in mice, since a bactericidal IgG3 did not elicit protection. In addition, no significant difference in protective capacity was observed with bactericidal and nonbactericidal IgG1 antibodies, indicating that bactericidal activity is not a requirement for protection mediated by certain anti-lipooligosaccharide A antibodies. A reduction in protection in C5-deficient mice was observed, suggesting a significant role for complement in certain host defense mechanisms against B. pertussis infection.

Mucosal immunization with filamentous hemagglutinin protects against Bordetella pertussis respiratory infection.

Mucosal immunization of mice with purified Bordetella pertussis filamentous hemagglutinin (FHA), by either the respiratory or the gut route, was found to protect against B. pertussis infection of the trachea and lungs. Intranasal immunization of BALB/c and (C57BL/6 x C3H/HeN)F1 adult female mice with FHA prior to B. pertussis aerosol challenge resulted in a 2 to 3 log reduction in number of bacteria recovered from the lungs and the tracheas of immunized mice in comparison to unimmunized controls. Intraduodenal immunization of adult mice with FHA before infection also resulted in approximately a 2 log reduction in the recovery of bacteria from the lungs and the tracheas of immunized mice in comparison to unimmunized controls. Immunoglobulin A and immunoglobulin G anti-FHA were both detected in bronchoalveolar lavage fluids of mucosally immunized mice. Limiting dilution analysis revealed a 60-fold increase in the frequency of FHA-specific B cells isolated from the lungs of mice immunized intranasally with FHA in comparison to unimmunized control mice. These data suggest that both gut and respiratory mucosal immunization with a major adhesin of B. pertussis generates a specific immune response in the respiratory tract that may serve as one means of mitigating subsequent B. pertussis respiratory infection.

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites.

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.

Active and passive immunization against Plasmodium yoelii sporozoites.

Three subunit vaccines based on the major repeat, (QGPGAP)n, and flanking regions of the Plasmodium yoelii circumsporozoite protein were designed, produced, and tested. All were immunogenic, but none gave consistent protection against a 40-200 sporozoite challenge. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of NYS1, an IgG3 MAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, indicating that the epitope on sporozoites recognized by this MAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by IFAT and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing QGPGAP but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not the absolute concentration, could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective MAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective MAbs and non-protective polyclonal antibodies will be important in the further development of malaria vaccines.

Characterization of Plasmodium yoelii monoclonal antibodies directed against stage-specific sporozoite antigens.

A battery of monoclonal antibodies against Plasmodium yoelii sporozoites was produced. Five of these (NYS1 through NYS5) were selected for characterization. All five were positive in the indirect immunofluorescent antibody test with P. yoelii sporozoites; however, each showed a different immunofluorescence pattern. Although NYS1 (immunoglobulin G3 [IgG3]), NYS2 (IgG3), and NYS3 (IgM) were positive in the circumsporozoite precipitation test, only NYS1 and NYS2 were able to neutralize sporozoite infectivity in mice. NYS4 (IgM) and NYS5 (IgG1) were not positive in the precipitation test and did not protect mice from sporozoite infection. All except NYS4 were species as well as stage specific. NYS4 cross-reacted with sporozoites of P. berghei. Electrophoretic immunoblotting analysis showed that these monoclonal antibodies detected sporozoite antigens of various molecular weights. Inhibition enzyme-linked immunosorbent assays indicated that each recognized a different antigenic epitope. The differences in their immunochemical and biological reactivity make them useful for screening a variety of P. yoelii antigens in recombinant DNA libraries. These antigens will be used in an animal model system for vaccine development.

Serological reactivity of in vitro cultured exoerythrocytic stages of Plasmodium berghei in indirect immunofluorescent or immunoperoxidase antibody tests.

Exoerythrocytic (EE) stages of Plasmodium berghei were cultivated in vitro in WI38 cells inoculated with sporozoites, and examined for serological reactivity by indirect immunofluorescent or immunoperoxidase tests. At 24 hours post-inoculation, sporozoite and red blood cell (RBC) stage antigens were equally distributed, but by 48 hours, RBC stage antigens predominated. Merozoites, produced by 72 hours, reacted strongly with anti-RBC stage sera, but were weakly reactive with anti-sporozoite sera. Hybridoma-produced monoclonal antibodies to the surface protective antigen of P. berghei sporozoites also reacted with 24- and 48-hour EE parasites, and with RR merozoites, suggesting that the sporozoite-protective antigen may also be synthesized by the EE stage. Extra-EE parasite antigens associated with the host cell nucleus were detected as early as 24 hours using anti-sporozoite and anti-RBC stage sera, but did not contain the sporozoite-protective antigen.

Production of monoclonal antibodies by hybridomas sensitized to sporozoites of Plasmodium berghei.

Hybridoma cell lines, which secreted antibodies directed against either the surface of Plasmodium berghei sporozoites or mosquito debris, were produced by fusion of spleen cells of P. berghei sporozoite-immunized mice with P3-X63-Ag8 myeloma cells. Four cloned antibody-secreting cell lines were successfully established. Two of these clones (F9 and G10) were obtained from the fusion of spleen cells from mice that had undergone two immunizations. These clones produced an IgM antibody that did not produce a CSP response on the sporozoite and did not neutralize the infectivity of the sporozoite in mice. One clone (B6), produced by the fusion of spleen cells from mice that had been given three immunizations, secreted an IgG antibody that did produce a CSP response on the sporozoite and neutralized the infectivity of the parasite. A fourth clone (D5) directed against mosquito debris was also found to produce an IgG antibody, but did not show a CSP reaction or neutralization of the sporozoite infectivity.

Malaria vaccination with irradiated sporozoites: serological evaluation of the antigen and antibody responses.

Vaccination against Plasmodium falciparum with attenuated sporozoites is the goal of the US Navy's Malaria Vaccine Program. One requirement in the development of this vaccine is an immunological test to study the sporozoite antigen and immune responses it induces. Using an indirect fluorescent antibody test (IFAT) and P. berghei in the mouse or rat as a model, we have made significant progress toward this goal. Four antigens were detected in vaccine preparations: sporozoite-specific antigens, mosquito antigens, antigens on the sporozoite that are common to erythrocytic stages, and bovine serum albumm, an antigenic element of the isolation medium no longer employed. The IFAT was a reliable monitor of vaccination in a mouse and rat model in conjunction with protection to challenge. The test was a sensitive monitor of vaccine quality. Anamnestic responses to bites of infected mosquitos were detected in mice previously immunized with irradiated sporozoites.