Variable Adjuvant Activity of Bordetella pertussis with Respect to the Primary and Secondary Immunization of Mice

We carried out a study on the adjuvant effect of Bordetella pertussis vaccine on the primary and secondary immune responses of the mouse to sheep erythrocytes, quantitating antibody-producing spleen cells and serum antibody. The simultaneous injection of sheep erythrocytes and B. pertussis, when compared to immunization with sheep red blood cells alone, resulted in an increased and prolonged multiplication of antibody-forming spleen cells. The adjuvant effect was also documented by increased production of serum hemolysins and agglutinins. Further, B. pertussis enhanced the priming effect of the antigen for the secondary response. However, when the bacterial adjuvant was given together with a second antigenic stimulus 41 days after the primary immunization, the peak values of direct and indirect plaque-forming spleen cells did not differ from the corresponding control animals further inoculated with sheep erythrocytes alone. Nonetheless, the influence of the bacterial adjuvant was still expressed by the delayed decrease of the numbers of plaque-forming spleen cells. On the basis of the X-Y-Z scheme it is suggested that B. pertussis cells as adjuvant enhance the multiplication of antigen-sensitive X cells or affect the initial stages of differentiation of these cells. This effect of the pertussis vaccine can be distinguished from a general proliferative action on other cells.     

Protection against autoimmune disease by bacterial agents. II. PPD and pertussis toxin as proteins active in protecting mice against experimental autoimmune encephalomyelitis

Bordetella pertussis and Mycobacterium tuberculosis, routinely used to promote the development of autoimmune disease, were recently reported to also be effective in inducing protection against an autoimmune disease. Thus, we previously demonstrated that SJL/J and (SJL/J x BALB/c)F₁ mice that are genetically susceptible to experimental autoimmune encephalomyelitis (EAE) become highly refractory to the induction of the disease following their exposure to B. pertussis and M. tuberculosis. In the present study, the pertussis toxin (PT) from B. pertussis and the purified protein derivative (PPD) of M. tuberculosis, were found to be sufficient to fully protect against EAE and thus may be the major bacterial components responsible for conferring protection. The 65-kDa heat-shock protein played only a marginal role in the protection against EAE induced by these bacteria. Both PT and PPD were protective when given before, but not after, the encephalitogenic challenge, and minute amounts (5–50 ng) emulsified in oil were sufficient to confer long-lasting resistance to EAE. The effect of PT or PPD on EAE differed from that of mitogens or bacterial superantigens, suggesting that their protection ability was not attributable merely to mitogenic or superantigenic properties. The mechanism of protection is not yet clear. Preliminary studies revealed a complex mechanism of protection whereby PPD and PT may operate differently. Thus, only PPD-induced, but not PT-induced, protection was transferrable by CD4⁺ T lymphocytes bearing an αγ Tcell antigen receptor. Neither PT nor PPD had a protective effect on EAE mediated by preformed pathogenic T lymphocytes and it is most likely that they exert their protection by affecting the development of such T lymphocytes. How bacteria such as B. pertussis and M. tuberculosis can either enhance the development of an autoimmune disease or protect against the disease is not yet clear. However, identifying PT and PPD as the bacterial components active in protection may allow a better understanding of the modulatory effects of bacteria and point to the potential use of such bacterial products in immunomodulation of autoimmune diseases.     

Time Relationships Between Injection of Antigen and Adjuvant I. Adjuvancy of Bordetella pertussis Given at Various Times Before the Primary Antigenic Stimulus

The adjuvant activity of Bordetella pertussis was investigated, both at the cellular and humoral levels, when the bacterial adjuvant was administered at various times before the primary antigenic stimulus of both 8 × 10⁶ and 4 × 10⁸ sheep erythrocytes. In all experiments, both adjuvant and erythrocyte antigen were given intraperitoneally. Adjuvant activity was measured on the basis of the primary immune response and on the degree of priming for the secondary immune reaction. A maximal effect on the primary immune response was found in mice which had received B. pertussis vaccine simultaneously with the erythrocyte antigen. A significant degree of adjuvancy was also demonstrable when B. pertussis vaccine was administered within a period of 48 hr before antigen. Adjuvant effectiveness was less as the time interval between the injection of antigen and adjuvant increased. The process of priming for the secondary response, however, was found to be equally affected when the adjuvant was given either simultaneously with the antigen or 12, 24, and 48 hr before sheep erythrocytes. On the basis of these and previous findings, it is suggested that adjuvancy of B. pertussis is caused chiefly by additional recruitment of responding cells. It is also suggested that memory-cell production may develop concurrently with, but independently of, the cellular proliferative events which lead to antibody formation during the primary immune response.     

The immune response suppressed by specific antibody

Homologous or heterologous anti-sheep erythrocyte serum given passively to normal rats markedly suppressed their spleen plaque-forming cell and serum antibody response to sheep erythrocytes. Passive immunization against bovine γ-globulin prevented `sensitization' by a first injection of the antigen. The suppressive effect of passive antibody was prevented or partially prevented by adjuvants, B. pertussis vaccine, S. typhi endotoxin or Freund's complete adjuvant. Passive antibody or adjuvants had relatively little effect on the primary response when given more than 24 hours after antigen or on the secondary response when given with antigen.

The kinetics of the early spleen plaque-forming cell response were measured using sheep erythrocytes as antigen, homologous anti-sheep erythrocyte serum for passive immunization and B. pertussis vaccine as adjuvant. With a constant antigen dose, larger amounts of passive antibody caused increased suppression. Suppression apparently resulted from a decrease in the number of cells initially responding; the rate of proliferation of cells that did respond was not affected by passive antibody. If the amount of passive antibody was kept constant, an increase in antigen dose or addition of adjuvant to the antigen increased the rate of proliferation of the cells that did respond; an effect sufficient to completely mask suppression produced by smaller amounts of passive antibody. These findings can be accounted for by assuming that passive antibody and higher antigen doses or adjuvant affect different interactions required for the antibody response. Thus, the magnitude of the antibody response is dependent not only on the amounts of antigen and passively given antibody but also on the amount and activity of any intentionally or unintentionally introduced factor having adjuvant activity.

     

Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.     

Accumulation of avirulent Bordetella pertussis Bvg mutants in the course of experimental whooping cough in mice

The duration and dynamics of accumulation of insertional Bordetella pertussis Bvg^? mutants were studied in the lungs of laboratory mice after intranasal and intravenous challenge by virulent bacteria of the causative agent of whooping cough. The ability of the virulent B. pertussis bacteria to remain for a long time in the body of mice was established. Using real-time PCR, a hundred genome equivalents of DNA of B. pertussis were detected in lungs of mice 2 months after infection regardless of type of challenge. Using the bacterial test bacteria were identified during only 4 weeks after challenge. Avirulent Bordetella pertussis Bvg^? mutants were accumulated in the period of infection. The percentage of the avirulent bacteria in the B. pertussis population reached 50% in 7–9 weeks after challenge. The obtained results show that laboratory mice can be used to study the dynamics of accumulation of B. pertussis insertion mutants in vivo and confirm the hypothesis that there is accumulation of insertion avirulent Bordetella pertussis Bvg^? mutants during development of pertussis infection in human.     

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Potentiated reagin response to egg albumin in Nippostrongylus brasiliensis infected rats: II. Time Course of The Reagin Response

The potentiated reagin response induced by infection with the nematode Nippostrongylus brasiliensis after sensitization with egg albumin and Bordetella pertussis vaccine was investigated to reveal the time course of the potentiated reagin response and the maximum reagin titre attained. The potentiated reagin response was found to be transitory but during this period high reagin titres to egg albumin were recorded.

Rat sensitized to egg albumin and treated with N. brasiliensis crude worm extract instead of an infection did not show a potentiated response.

     

Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen‐induced immunity and prevents allergic pulmonary pathology in a murine model

Background Virulent Bordetella pertussis, the causative agent of whooping cough, exacerbates allergic airway inflammation in a murine model of ovalbumin (OVA) sensitization. A live genetically attenuated B. pertussis mucosal vaccine, BPZE1, has been developed that evokes full protection against virulent challenge in mice but the effect of this attenuated strain on the development of allergic responses is unknown. Objective To assess the influence of attenuated B. pertussis BPZE1 on OVA priming in a murine model of allergic airway inflammation. Methods Mice were challenged with virulent or attenuated strains of B. pertussis, and sensitized to allergen (OVA) at the peak of bacterial carriage. Subsequently, airway pathology, local inflammation and OVA‐specific immunity were examined. Results In contrast to virulent B. pertussis, live BPZE1 did not exacerbate but reduced the airway pathology associated with allergen sensitization. BPZE1 immunization before allergen sensitization did not have an adjuvant effect on allergen specific IgE but resulted in a statistically significant decrease in airway inflammation in tissue and bronchoalveolar lavage fluid. BPZE1 significantly reduced the levels of OVA‐driven IL‐4, IL‐5 and IL‐13 but induced a significant increase in IFN‐γ in response to OVA re‐stimulation. Conclusions These data demonstrate that, unlike virulent strains, the candidate attenuated B. pertussis vaccine BPZE1 does not exacerbate allergen‐driven airway pathology. BPZE1 may represent an attractive T‐helper type 1 promoting vaccine candidate for eradication of whooping cough that is unlikely to promote atopic disease.     

Sustained protective immunity against Bordetella pertussis nasal colonization by intranasal immunization with a vaccine-adjuvant combination that induces IL-17-secreting TRM cells

Current acellular pertussis (aP) vaccines induce strong antibody and Th2 responses but fail to protect against nasal colonization and transmission of Bordetella pertussis. Furthermore, immunity wanes rapidly after immunization. We have developed a novel adjuvant combination (called LP-GMP), comprising c-di-GMP, an intracellular receptor stimulator of interferon genes (STING) agonist, and LP1569, a TLR2 agonist from B. pertussis, which synergistically induces production of IFN-β, IL-12 and IL-23, and maturation of dendritic cells. Parenteral immunization of mice with an experimental aP vaccine formulated with LP-GMP promoted Th1 and Th17 responses and conferred protection against lung infection with B. pertussis. Intranasal immunization with the same aP vaccine-induced potent B. pertussis-specific Th17 responses and IL-17-secreting respiratory tissue-resident memory (T_RM) CD4 T cells, and conferred a high level of protection against nasal colonization as well as lung infection, which was sustained for at least 10 months. Furthermore, long-term protection against nasal colonization with B. pertussis correlated with the number of IL-17-secreting T_RM cells in nasal tissue. Our study has identified an approach for inducing IL-17-secreting T_RM cells that sustain sterilizing immunity against nasal colonization of mice with B. pertussis, and could form the basis of a third generation pertussis vaccine for humans.     

The immunological response of the rat to infection with Taenia taeniaeformis: VII. Immunization by oral and parenteral administration of antigens

Rats immunized with in vitro products and saline-soluble antigens derived from Taenia taeniaeformis were found to be significantly protected against challenge infection. Oral and intraperitoneal administration of antigen solutions alone were effective in stimulating resistance. Adjuvants, however, were required for successful immunization when the antigens were injected intramuscularly. Bordetella pertussis and aluminium hydroxide were able to improve markedly the protective effects of antigens given parenterally by either route, but Freund's complete adjuvant (FCA) was not effective as an adjuvant in this system. Reaginic antibodies to parasite antigens were detected in the sera of rats immunized with parasite antigens and B. pertussis or Al(OH)₃, but none were detected in those given antigens incorporated in FCA. The possible role of reaginic antibodies in immunity to T. taeniaeformis is discussed.

A single dose of antigen given orally produced significant protection. Increasing the number of daily doses of antigen administered orally enhanced the degree of protection to a limited but significant extent. There did not appear, however, to be any advantage to giving large doses (> 1 mg protein) of antigen, or extending the immunizing schedule over several weeks. Reaginic antibodies were not detected in the sera of rats immunized orally, but these animals were resistant to both oral and intravenous challenge infection with parasites. These observations are discussed in relation to the phenomena of immune exclusion of antigen by the gut, and gastrointestinally induced systemic tolerance with respect to IgE production.

Sera from rats immunized by all routes were found to be ineffective in conferring resistance upon recipients when given at the dose of 1 ml/rat. Furthermore, sera from donors vaccinated intramuscularly with saline soluble antigens and B. pertussis increased the susceptibility of recipient rats to infection with T. taeniaeformis. This is in sharp contrast to our previous experience in which we have shown that sera from rats with an active infection are highly effective in passive transfer. Possible reasons for these observations are discussed. The requirements for adequately controlled immunization procedures to assess the contributory effects of adjuvant type and the route of antigen inoculation in immunizing against taeniid infections are emphasized in the discussion.

     

Stimulation of non-specific resistance by heterologous endotoxins and experimental immunity to Bordetella pertussis in mice

A single intraperitoneal administration of Bordetella pertussis vaccine produced within a few days an increased resistance in mice against intracerebral infection with B. pertussis strain 18–323 such as has previously been described by Evans and Perkins as early or interference immunity.

Intraperitoneal administration of the endotoxin of B. pertussis induced a relatively transient resistance against intracerebral infection with Salmonella typhi strain Ty2, but not against intracerebral infection with B. pertussis organisms.

When the treatment was made intracerebrally however, heterologous and homologous endotoxins as well as a synthetic double-stranded RNA complex of polyriboinosinic and polyribocytidylic acids (poly I.C) could increase the resistance of mice against intracerebral infection with B. pertussis organisms. In brains of animals thus treated, evident suppression of bacterial growth comparable to that in a passive immunity experiment was seen.

By the use of brain extract prepared from mice or rats treated intracerebrally with heterologous endotoxin, the non-specific resistance could be successfully induced in mice.

To substantiate any possible relation of such a non-specific resistance induced by endotoxins to the early immunity seen after the intraperitoneal injection of B. pertussis vaccine further efforts are necessary.

     

Antibody response to a protein antigen (ovalbumin) in dissociated spleen cell cultures from primed mice: Evidence for a suppressive effect of antigen

A system is described with which an antibody response to soluble chicken ovalbumin can be obtained in dissociated spleen cell cultures from primed mice. This was assayed by determining specific anti-ovalbumin plaque-forming cells (PFC). Only IgG-producing cells (indirect PFC) were seen.

The mice received alum-precipitated ovalbumin i.p. and cultures were set up either late in the primary splenic response (23–27 days after injection) or near the peak of the response (10–14 days). With both groups secondary responses were seen in the presence of antigen on the fourth day of culture. In the second group PFC were present in the cultures at a high level initially. The number of PFC decreased over the first 2 days, the rate of decrease being greater in the presence of antigen.

When mice were primed with alum-precipitated ovalbumin together with B. pertussis as adjuvant, responses could not now be obtained in culture. With mice taken late in the primary response (23–27 days), a considerable number of PFC were present in the cultures initially. The number decreased throughout the 4-day period and the decline was accelerated by antigen. With mice taken near the peak of the response (10–14 days) no PFC were seen in culture, even on the first day, in the presence or absence of antigen. The first phenomenon, suppression by antigen, is considered to be of general significance since it may also be seen in cultures from mice receiving no B. pertussis as described. It is suggested that the second phenomenon is due to adverse culture conditions resulting from the white cell mobilization initiated by B. pertussis; after 23–27 days conditions have recovered to a point where suppression but not the secondary response can occur. The suppressive effect of antigen could be related to antigenic competition or tolerance.

     

Plasmacytoid dendritic cell-derived IFNα modulates Th17 differentiation during early Bordetella pertussis infection in mice

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis). T helper 17 (Th17) cells have a central role in the resolution of the infection. Emerging studies document that type I interferons (IFNs) suppress Th17 differentiation and interleukin (IL)-17 responses in models of infection and chronic inflammation. As plasmacytoid dendritic cells (pDCs) are a major source of type I IFNs, we hypothesize that during B. pertussis infection in mice, pDC-derived IFNα inhibits a rapid increase in Th17 cells. We found that IFNα-secreting pDCs appear in the lungs during the early stages of infection, while a robust rise of Th17 cells in the lungs is detected at 15 days post-infection or later. The presence of IFNα led to reduced Th17 differentiation and proliferation in vitro. Furthermore, in vivo blocking of IFNα produced by pDCs during infection with B. pertussis infection resulted in early increase of Th17 frequency, inflammation, and reduced bacterial loads in the airways of infected mice. Taken together, the experiments reported here describe an inhibitory role for pDCs and pDC-derived IFNα in modulating Th17 responses during the early stages of B. pertussis infection, which may explain the prolonged nature of whooping cough.     

A critical role for the TLR signaling adapter Mal in alveolar macrophage-mediated protection against Bordetella pertussis

Bordetella pertussis causes whooping cough, an infectious disease of the respiratory tract that is re-emerging despite high vaccine coverage. Here we examined the role of Toll-like receptor (TLR) adapter protein Mal in the control of B. pertussis infection in the lungs. We found that B. pertussis bacterial load in the lungs of Mal-defective (Mal^−/−) mice exceeded that of wild-type (WT) mice by up to 100-fold and bacteria disseminated to the liver in Mal^−/− mice and 50% of these mice died from the infection. Macrophages from Mal^−/− mice were defective in an early burst of pro-inflammatory cytokine production and in their ability to kill or constrain intracellular growth of B. pertussis. Importantly, the B. pertussis bacterial load in the lungs inversely correlated with the number of alveolar macrophages. Despite the maintenance and expansion of other cell populations, alveolar macrophages were completely depleted from the lungs of infected Mal^−/− mice, but not from infected WT mice. Our findings define for the first time a role for a microbial pattern-recognition pathway in the survival of alveolar macrophages and uncover a mechanism of macrophage-mediated immunity to B. pertussis in which Mal controls intracellular survival and dissemination of bacteria from the lungs.     

Macrophage behaviour during the complaisant phase of murine pertussis

During the complaisant phase of murine pertussis, Bordetella pertussis survived within lung macrophages. Bactericidal activity in the complaisant mouse, which was shown to be activated by viable superinfection, could also be elicited by alcohol killed B. pertussis. Isolated complaisant cells in vitro, although not superior to normal cells in phagocytosis, showed an enhanced antibacterial activity against large numbers of viable B. pertussis. The relationship of cytophilic antibody to this enhanced bactericidal activity is unknown, but circulating protective antibody was relatively unimportant at this stage of infection. Although a higher titre of opsonins was found in complaisant mice, opsonization with optimal quantities of normal or complaisant phase serum neither increased phagocytosis nor affected the outcome of intracellular digestion.     

Evaluation of Level of Agreement in Bordetella Species Identification in Three U.S. Laboratories during a Period of Increased Pertussis

While PCR is the most common method used for detecting Bordetella pertussis in the United States, most laboratories use insertion sequence 481 (IS481), which is not specific for B. pertussis; therefore, the relative contribution of other Bordetella species is not understood. The objectives of this study were to evaluate the proportion of other Bordetella species misidentified as B. pertussis during a period of increased pertussis incidence, determine the level of agreement in Bordetella species detection between U.S. commercial laboratories and the CDC, and assess the relative diagnostic sensitivity of CDC''s PCR assay when using a different PCR master mix. Specimens collected between May 2012 and May 2013 were tested at two U.S. commercial laboratories for B. pertussis and B. parapertussis detection. Every fifth specimen positive for IS481 and/or IS1001 with cycle threshold (C_T) values of ≤35 was sent to CDC for PCR testing that identifies Bordetella species. Specimens with indeterminate or negative results in the CDC PCR were tested using an alternate PCR master mix. Of 755 specimens, there was agreement in species identification for 83.4% (n = 630). Of the specimens with different identifications (n = 125), 79.2% (n = 99) were identified as indeterminate B. pertussis at CDC. Overall, 0.66% (n = 5) of the specimens were identified as B. holmesii or B. bronchiseptica at CDC. Of 115 specimens with indeterminate or negative results, 46.1% (n = 53) were B. pertussis positive when tested by an alternate master mix, suggesting a possible increase in assay sensitivity. This study demonstrates good agreement between the two U.S. commercial laboratories and CDC and little misidentification of Bordetella species during the 2012 U.S. epidemic.     

Vag8, a Bordetella pertussis bvg-Regulated Protein

Bordetella pertussis expresses a bvg-regulated 95-kDa protein, Vag8, encoded by vag-8. Southern blot analysis indicates that strains of Bordetella bronchiseptica and Bordetella parapertussis have DNA homologous to vag-8. Antiserum raised to a fusion of maltose binding protein to an N-terminal 60-kDa fragment of Vag8 recognizes the native 95-kDa protein in immunoblots of B. pertussis and B. bronchiseptica but not B. parapertussis. A 95-kDa protein-negative derivative of B. pertussis 18323 containing a deletion of vag-8 colonized mice as efficiently as the parent B. pertussis strain in a mouse aerosol model of pertussis.     

A Murine Model in Which Protection Correlates with Pertussis Vaccine Efficacy in Children Reveals Complementary Roles for Humoral and Cell-Mediated Immunity in Protection against Bordetella pertussis

The results of phase 3 efficacy trials have shown that acellular and whole-cell pertussis vaccines can confer protection against whooping cough. However, despite the advances in vaccine development, clinical trials have not provided significant new information on the mechanism of protective immunity against Bordetella pertussis. Classical approaches based on measurement of antibody responses to individual antigens failed to define an immunological correlate of protection. A reliable animal model, predictive of acellular and whole-cell pertussis vaccine potency in children, would facilitate an elucidation of the mechanism of immune protection against B. pertussis and would assist in the regulatory control and future development of pertussis vaccines. In this study, we have shown that the rate of B. pertussis clearance following respiratory challenge of immunized mice correlated with vaccine efficacy in children. Using this model together with mice with targeted disruptions of the gamma interferon (IFN-γ) receptor, interleukin-4 or immunoglobulin heavy-chain genes, we have demonstrated an absolute requirement for B cells or their products in bacterial clearance and a role for IFN-γ in immunity generated by previous infection or immunization with the whole-cell pertussis vaccine. The results of passive immunization experiments suggested that protection early after immunization with acellular pertussis vaccines is mediated by antibody against multiple protective antigens. In contrast, more complete protection conferred by previous infection or immunization with whole-cell pertussis vaccines reflected the induction of Th1 cells. Our findings suggest that the mechanism of immunity against B. pertussis involves humoral and cellular immune responses which are not directed against a single protective antigen and thus provide an explanation for previous failures to define an immunological correlate of protection.