Mechanism of adjuvant activity of poly A:U on antibody responses to hapten-carrier conjugates

The adjuvant action of poly A:U has been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. Administration of poly A:U at the time of primary immunization with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) shortens the induction period for, and heightens the magnitude of peak anti-DNP antibody and specific memory cell production. In order to define the cellular locus of poly A:U action, the effect of this adjuvant on adoptive secondary anti-DNP antibody responses was studied. Spleen cells from DNP-KLH-primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine gamma globulin (BGG), can be stimulated to do so when an appropriate dose of poly A:U is administered with DNP-BGG. The capacity for poly A:U to exert this effect requires the presence of T lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro prior to adoptive transfer abrogates the response to DNP-BGG plus poly A:U. Moreover, evidence is presented that demonstrates that poly A:U exerts its adjuvant action on the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of poly A:U to augment adoptive secondary anti-DNP responses to the DNP derivative of a nonimmunogenic copolymer of d-glutamic acid and d-lysine (d-GL) for which there are few or no specific, functional T cells.     

Adjuvant actions of polyclonal lymphocyte activators. II. Comparison and characterization of their actions in initiation and potentiation of immune responses to T-dependent and T-independent soluble antigens

Various polyclonal lymphocyte activators (PLA) such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide of Escherichia coli (LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and polyadenylic-polycytidylic acid (poly A:U) were compared in their effects on antibody response to T-dependent antigen (bovine gamma globulin (BGG) and dinitrophenylated (DNP)-BGG) and T-independent antigen (DNP-Ficoll) and on induction of tolerance to T-dependent antigen. All of these PLA acted more or less to trigger the initiation of the antibody-forming mechanism for deaggregated BGG (DBGG) or DNP-BGG through their actions on the carrier-specific T-cell function. All of these PLA tested also acted more or less to inhibit the induction of the carrier-specific T-cell tolerance to DBGG. Moreover, some of these PLA could act to augment antibody response to DNP-Ficoll. The adjuvant action of PLA in the response to DNP-Ficoll worked as well in athymic nu/nu mice as in nu/+ mice, whereas that in the response to DNP-BGG did not occur in athymic nu/nu mice. The order of the strength of the action of PLA to trigger the initiation of the whole immune response to DBGG, that to trigger the carrier-specific T-cell function to DNP-BGG, and that to inhibit the induction of the whole tolerance to DBGG was very similar to each other: i.e., CPS-K ? Con A > LPS, DS, poly A:U, PWM and PHA. By contrast, the order of the strength of the action to inhibit the induction of T-cell tolerance to DBGG was ≧ = LPS > Con A, PWM and poly A:U > DS and PHA, and that of the action to augment the antibody response to DNP-Ficoll was CPS-K > LPS > Con A. CPS-K was the most potent in all of these immunological activities. It was concluded that PLA act generally to stimulate the immune response at its initiation step in which T cells in the case of T-dependent antigen and B cells in the case of T-independent antigen play a predominant role, but that individual PLA share this adjuvant activity in different fashions.     

In vitro correction of antigen-induced immune suppression: effects of poly(A) poly(U) and prostaglandin E

Incubation of SJL or DBA/1 mouse spleen cells with poly(lTyr, lGlu)-polylPro—polylLys, (T, G)-Pro—L in vitro reduced the immune response potential of the cells to this immunogen as tested by adoptive transfer into irradiated, syngeneic recipients, followed by immunization with (T, G)-Pro—L in complete Freund's adjuvant. This reduction in immunocompetence was antigen-specific, since incubation with another antigen (rabbit immunoglobulin G) did not result in a suppression of responsiveness of the cells to subsequent in vivo immunization with (T, G)-Pro—L. Incubation of the spleen cell-(T, G)-Pro—L mixture in the presence of either prostaglandin E₁(PGE₁) or polyadenylic-polyuridylic acid (poly(A)·poly (U)) restored the immune response potential to the normal level. Incubation of (T, G)-Pro—L with spleen cells had no effect on cyclic AMP accumulation, whereas incubation of PGE₁ with the cells stimulated cyclic AMP production, irrespective of the presence of antigens. In contrast, the level of cyclic AMP was not affected by poly(A) · poly(U). The difference in cyclic AMP accumulation suggests that PGE₁ and poly(A) · poly(A) modify immune responsiveness by different mechanisms. The above observations were verified both in SJL and DBA/1 mice, which are the respective genetic high and low responders to (T, G) -Pro—L. This implies that the modifications of responsiveness described are not related to the genetic control of immune response to this immunogen.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

Influence of poly A-poly U on early events in the immune response in vitro

Adjuvant action of artificial homopolyribonucleotides and nucleic acid digests was studied in mouse spleen-cell cultures, stimulated with sheep red cells by the technique of Mishell and Dutton. Assays of antibody-forming cells were performed according to the Jerne and Nordin technique. Doublestranded poly A-poly U, and RNA digests had adjuvant activity over a wide dose range (0.01–100 μg/ml). DNA digests, single stranded polynucleotides (poly A, poly U, poly C) and mixtures of ribonucleosides and ribonucleotides had little or no enhancing effect at comparable doses. From our study of the kinetics of increase of plaque-forming cells in cultures treated with poly A-poly U, in conjunction with in vivo studies of others, we concluded that adjuvant action, during induction, indirectly increased the number of bone marrow-derived cells that responded to antigenic stimulation by increasing the effectiveness of the thymus-derived cells in the culture.     

Regulation of the immune system by synthetic polynucleotides. VII. Suppression induced by pretreatment with poly A:U.

Pretreatment of mouse spleen cells with polyadenylic-polyuridylic acid complexes (poly A:U) either in vivo or in vitro 24 hr prior to addition of antigen, resulted in a substantial time dependent decrease in anti-SRBC PFC. Enhancement was observed 6 hr after poly A:U, while inhibition did not become evident until 24 hr after pretreatment. Inhibition of the PFC response appeared to result from poly A:U activation of a nylon wool adherent, T suppressor cell, capable of diminishing the response of normal spleen cells exposed to antigen on co-culture.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Enhanced anti-tumor effects of HPV16E749–57-based vaccine by combined immunization with poly(I:C) and oxygen-regulated protein 150

Background: It is well known that both heat shock protein (HSP) and Toll-like receptor (TLR)3 agonist polyinosinic:polycytidylic acid (poly(I:C)) are capable of promoting the antigen-specific immune responses. In the current study, we assessed whether the anti-tumor effects of the HPV16E7_49–57-based vaccine can be elevated by combined applications of poly(I:C) and oxygen-regulated protein 150 (ORP150) in a mouse cervical cancer model. Methods: Recombinant mouse ORP150 and HPV E7_49–57 peptide were combined to passively form the ORP150–E7_49–57 complex under heat shock conditions. The effects of ORP150–E7_49–57 complex plus poly(I:C) adjuvant on lymphocyte proliferation and functional cytotoxic T cells were investigated by methyl thiazolyl tetrazolium (MTT), ELISPOT, and non-radioactive cytotoxicity assays. Finally, the complex's therapeutic anti-tumor effects with and without adjuvant therapy were observed in a tumor challenge experiment. Results: This combination vaccine approach significantly enhanced the proliferation of splenocytes and induced strong E7_49–57-speci?c CTL responses. More importantly, the ORP150–E7_49–57 complex plus poly(I:C) vaccine format demonstrated more potent anti-tumor effects than ORP150–E7_49–57 complex alone or E7_49–57 plus poly(I:C) in TC-1 tumor-bearing mice. Conclusion: Both poly(I:C) and ORP150 chaperone can synergistically enhance the anti-tumor effects of the HPV16E7_49–57-based vaccine in vitro and in vivo. This strategy provides a platform for the design of a tumor therapeutic vaccine capable of inducing an effective anti-tumor immune response.     

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer. Received: 27 August 1997 / Accepted: 24 April 1998     

The Adjuvant Double Mutant Escherichia coli Heat Labile Toxin Enhances IL-17A Production in Human T Cells Specific for Bacterial Vaccine Antigens

The strong adjuvant activity and low enterotoxicity of the novel mucosal adjuvant double mutant Escherichia coli heat labile toxin, LT(R192G/L211A) or dmLT, demonstrated in mice, makes this molecule a promising adjuvant candidate. However, little is known about the mechanisms responsible for the adjuvant effect of dmLT or whether dmLT also has an adjuvant function in humans.We investigated the effect of dmLT on human T cell responses to different bacterial vaccine antigens: the mycobacterial purified protein derivative (PPD) antigen, tested in individuals previously vaccinated with Bacillus Calmette-Guérin, the LT binding subunit (LTB), evaluated in subjects immunised with oral inactivated whole cell vaccines against enterotoxigenic Escherichia coli, and Streptococcus pneumoniae whole cell vaccine antigens, tested in subjects naturally exposed to pneumococci. We found that dmLT enhanced the production of IL-17A by peripheral blood mononuclear cells in response to all antigens tested. dmLT had comparable effects on IL-17A responses to PPD as the single mutant LT(R192G) adjuvant, which has demonstrated clinical adjuvant activity in humans. Neutralisation of IL-1β and IL-23, but not IL-6, suppressed the IL-17A-enhancing effect of dmLT. Furthermore, CD4+ T cells produced higher levels of IL-17A when stimulated with monocytes pulsed with PPD and dmLT compared to PPD alone, supporting an important role of antigen presenting cells in enhancing IL-17A responses. dmLT also potentiated mitogen-induced IL-17A and IL-13 production. However, dmLT had variable influences on IFN-γ responses to the different stimuli tested.Our demonstration of a potent ability of dmLT to enhance human Th17 type T cell responses to bacterial vaccine antigens encourages further evaluation of the adjuvant function of dmLT in humans.     

Experimental allergic encephalomyelitis-inducing activity of synthetic polyadenylic and polyuridylic homopolymers and complexes in guinea pigs.

In addition to the capacity of polyuridylic acid (poly(U)) or complexes of polyadenylic acid (poly(A)) and Poly(U) (poly(A : U)) to serve as adjuvants for induction of experimental allergic encephalomyelitis (EAE) in guinea pigs sensitized to spinal cord or myelin basic protein, these synthetic polynucleotide homopolymers possess inherent EAE-inducing activity for this host. EAE activity of poly (A) or poly(A : U) was demonstrable following a single injection of the purine homopolymer or the complex in incomplete Freund's adjuvant (IFA). EAE-inducing activity of poly(U) was observed only in guinea pigs initially primed with this pyrimidine homopolymer in IFA.     

Increased Antigen Specific T Cell Numbers in the Absence of Altered Migration or Division Rates as a Result of Mucosal Cholera Toxin Administration

Cholera toxin (CT) is a mucosal adjuvant capable of inducing strong immune responses to co-administered antigens following oral or intranasal immunization of mice. To date, the direct effect of CT on antigen-specific CD4⁺ T cell migration and proliferation profiles in vivo is not well characterized. In this study, the effect of CT on the migration pattern and proliferative responses of adoptively transferred, CD4⁺ TCR transgenic T cells in orally or intranasally vaccinated mice, was analyzed by flow cytometry. GFP-expressing or CFSE-labeled OT-II lymphocytes were adoptively transferred to naïve C57BL/6 mice, and mice were subsequently vaccinated with OVA with or without CT via the oral or intranasal route. CT did not alter the migration pattern of antigen-specific T cells, regardless of the route of immunization, but increased the number of transgenic CD4⁺ T cells in draining lymphoid tissue. This increase in the number of transgenic CD4⁺ T cells was not due to cells undergoing more rounds of cellular division in vivo, suggesting that CT may exert an indirect adjuvant effect on CD4⁺ T cells. The findings reported here suggest that CT functions as a mucosal adjuvant by increasing the number of antigen specific CD4⁺ T cells independent of their migration pattern or kinetics of cellular division.     

Immune Responses to HBsAg Conjugated to Protein D of Non-Typeable Haemophilus influenzae in Mice

BackgroundHepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1–6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten–carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine.ConclusionsRecombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.     

A unidirectional carrier effect

A surprising unidirectional carrier effect has been observed in the antibody response to myoglobin-ferritin conjugate. This conjugate serves as a hapten-carrier complex for myoglobin-specific T cells to help ferritin-specific B cells make anti-ferritin antibodies, but it does not function for ferritin-specific T cells to help myoglobin-specific B cells to make anti-myoglobin. Therefore, myoglobin-ferritin does not bypass the Ir gene defect of low responders to myoglobin. In contrast, myoglobin-fowl γ-globulin does induce anti-myoglobin antibodies in low responder mice and thus bypasses the Ir gene defect. Both complexes are covalently coupled. Since the myoglobin-ferritin conjugate serves for myoglobin-specific T cells to help myoglobin-specific B cells, the myoglobin in the conjugate is not altered in a way that would prevent recognition by myoglobin-specific B cells. Similarly, the conjugate serves for ferritin-specific helper T cells to help ferritin-specific B cells, so it can be recognized functionally by ferritin-specific T helper cells. Explanations such as unidirectional induction of or sensitivity to bystander help, or T-cell suppression, have been excluded. While the explanation for this unexpected observation is not yet certain, several possibilities are discussed to explain this novel phenomenon, which is believed to be the first example of such a unidirectional carrier effect between two proteins.     

Immunologic-mediated Protection of Trypanosoma congolense-Infected Mice by Polyribonucleotides

SYNOPSIS. When the synthetic polyribonucleotides polyinosinic acid-polycytidylic acid (poly I poly C) and polyadenylic acid-polyuridylic acid (poly A poly U) were tested against mice infected with varying numbers of Trypanosoma congolense the results varied with the method of passage of trypanosomes in mice. Thus, when 100 flagellates were passaged every 7th day and experiments were initiated with these trypanosomes from mice on the 7th day of their infection, the protective effects of poly I poly C and poly A poly U apparently varied independently of each other as assayed by the mean parasitemias and cumulative mortalities of infected mice. Poly I poly C-resistant and poly I poly C-susceptible variants (“R” and “S”, respectively) were isolated and maintained in mice by passage of 10⁶ trypanosomes every 4th day. Mice infected with these variants responded consistently to poly I poly C and poly A poly U injections in that mice infected with the “R” variant showed no response to either polyribonucleotide but those infected with the “S” variant consistently had a decrease in mean parasitemias and cumulative mortality when treated with poly I poly C, but not with poly A poly U. Using mice infected with the “S” variant, the protective effect of poly I poly C was dose-dependent and best protection was afforded when 1st injections of poly I poly C were given around the time of infection of the mice. The protective effects of the synthetic polyribonucleotides used in these experiments are probably due to their immunologic enhancing capacities and not to interferon. To support this, injections of Newcastle disease virus, a strong inducer of interferon in mice, did not protect against T. congolense in mice. It was not possible to determine whether serum from poly I poly C-treated mice had a greater neutralizing effect upon trypanosomes in vitro than serum from saline-treated mice since any effect of antibody was masked by a naturally occurring inhibitor in normal mouse serum which was reduced during infection. The protective effects of poly I poly C in T. congolense-infected mice were reversed by treatment with cyclophosphamide. This strong immunosuppressant, however, could not reverse the protective effects of poly I poly C against mice infected with Semliki Forest virus, strongly suggesting that the protective mechanisms stimulated by poly I poly C in these 2 infections were different. The response of mice infected with the “R” and “S” variants of T. congolense to synthetic polyribonucleotides is discussed in relation to antigenic variation of trypanosomes.     

Non-snRNP U1A levels decrease during mammalian B-cell differentiation and release the IgM secretory poly(A) site from repression

A regulated shift from the production of membrane to secretory forms of Immunoglobulin M (IgM) mRNA occurs during B cell differentiation due to the activation of an upstream secretory poly(A) site. U1A plays a key role in inhibiting the expression of the secretory poly(A) site by inhibiting both cleavage at the poly(A) site and subsequent poly(A) tail addition. However, how the inhibitory effect of U1A is alleviated in differentiated cells, which express the secretory poly(A) site, is not known. Using B cell lines representing different stages of B cell differentiation, we show that the amount of U1A available to inhibit the secretory poly(A) site is reduced in differentiated cells. Undifferentiated B cells have more total U1A than differentiated cells and a greater proportion of this is not associated with the U1snRNP. We show that this is available to inhibit poly(A) addition at the secretory poly(A) site using cold competitor RNA oligos to de-repress poly(A) addition in nuclear extracts from the respective cell lines. In addition, endogenous non-snRNP associated U1A-immunopurified from the different cell lines-inhibits poly(A) polymerase activity proportional to U1A recovered, suggesting that available U1A level alone is responsible for changes in its inhibitory effect at the secretory IgM poly (A) site.     

Polyinosinic-polycytidylic acid limits tumor outgrowth in a mouse model of metastatic lung cancer

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c(+)CD8(+)), NKT cells, CD8(+) T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.     

Studies on delayed hypersensitivity to protein antigen. Antigen-specific suppression of delayed hypersensitivity by chemically modified antigen.

An extensively modified protein antigen (methylated bacterial α-amylase, M-BαA) which was neither reactive with anti-BαA antibody nor able to induce a humoral anti-BαA response, retained the ability to prime native BαA-specific T cells which were responsible for the enhanced anti-BαA response to subsequent immunization with BαA and delayed hypersensitivity (DH). The splenic T cell-rich fraction from mice primed with M-BαA collaborated with a native BαA-primed B cell-rich fraction to give a good adoptive IgG anti-BαA response in syngeneic irradiated mice, whereas M-BαA-primed B cell fractions failed to cooperate with native BαA-primed T cell fractions. Splenic T cells from mice given a subcutaneous (s.c.) injection of M-BαA in complete Freund's adjuvant (CFA) exhibited DH in syngeneic cyclophosphamide-treated mice. In the present study, native and methylated BαA were tested for their ability to generate suppressor T cells capable of inhibiting the development of DH. An intraperitoneal (i.p.) injection of either native or methylated BαA in incomplete Freund's adjuvant (IFA) interferred with the development of DH to M-BαA by an s.c. injection of the same antigen in CFA. Transfer of spleen cells from mice given an i.p. injection of either of these antigens 5 days previously, suppressed antigen-specifically induction and expression of DH in the syngeneic recipient mice. The suppressive activity was sensitive to treatment with anti-θ antiserum plus complement. These results indicate that the early phase of inhibition of DH after an i.p. injection is in part mediated by suppressor T cells and that M-BαA cross-reacts with native BαA at the suppressor T cell level as well as the level of effector T cells in DH.     

Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia

Background

Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant.

Methodology/Principal Findings

Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas.

Conclusions

These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.     

Unresponsiveness of MRL/MP-lpr/lpr mice to antigen given subcutaneously in adjuvant: partial restoration of response after local injection of B cells

MRL/lpr mice were used as a model for seeking information on the role of B cells as antigen-presenting cells in vivo. In confirmation of the finding of other workers, MRL/lpr mice with pronounced lymphadenopathy failed to undergo T cell priming in lymph nodes (LN) after s.c. injection of keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA): thus, in contrast to normal mice or young MRL/lpr mice, the LN cells from older MRL/lpr mice failed to give secondary T proliferative responses to the injected antigen in vitro. Because previous work from this laboratory has shown that B cells play a major role in priming T cells in LN of normal mice, we tested the possibility that the lack of T cell priming to KLH seen in older MRL/lpr LN reflected the relative paucity of B cells. To test this idea, MRL/LPR mice were injected s.c. with B cells taken from normal or young MRL/lpr mice 1 day before priming with KLH/CFA. In the case of old (20 to 24 wk) MRL/lpr mice with massive lymphadenopathy, prior injection of B cells had virtually no effect in promoting LN priming. In marked contrast, injection of B cells into mice exhibiting less-pronounced LN enlargement (mice tested at 14 to 16 wk) was highly effective in restoring LN priming; before B cell injection, the LN of these mice contained only 2 to 5% B cells. These findings add to the evidence that T cell priming in LN is heavily dependent on the presence of B cells.