A proposal for safety standards for human use of cholera toxin (or Escherichia coli heat-labile enterotoxin) derivatives as an adjuvant of nasal inactivated influenza vaccine.

Cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) are not only the causative agents of diarrhea but are also strong mucosal adjuvants which enhance immune responses to mucosally coadministered bystander antigens. One of the most promising applications of these toxins would be as mucosal adjuvant of nasal influenza vaccine. In comparison to current inactivated vaccines, the nasal vaccine provides superior cross-protection by inducing production of cross-reacting anti-viral IgA antibodies in the respiratory tract even when the vaccine strain is different from the epidemic strain. On the use of the toxins as mucosal adjuvants in humans, toxicity and allergenicity of the toxins are problems which impinge on safety. To resolve these problems, various approaches have been attempted to produce less toxic and less allergenic CT (or LT) derivatives. We now propose the following standards for human use of safer CT (or LT) derivatives as an adjuvant of a nasal influenza vaccine. Thus, CT (or LT) derivatives can be administered intranasally together with a current inactivated influenza vaccine, provided they meet the following criteria. 1) A single dose of the derivatives, administered intranasally by spraying, should be around 100 Eg/adult in a volume of less than 0.5 ml. 2) CT (or LT) derivatives should retain the properties of the native CT (or LT), i. e., the ability to augment secretory IgA and serum IgG Ab responses to viral surface glycoproteins, when administered intranasally together with an inactivated influenza vaccine. 3) CT (or LT) derivatives should not induce IgE Ab responses to the vaccine, as well as to the CT (or LT) itself. 4) The CT (or LT) should be nontoxic; the toxicity of the derivatives, as determined by the Y-1 adrenal cell assay, should not exceed 1/100 EC(50) of the native CT (or 1/1000 ECi of the native CT). 5) CT (or LT) derivatives should not cause serious disease in guinea pigs when administered intranasally or intraperitoneally at the dose used in humans (around 100 Eg).

Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin).

Inactivated influenza vaccine was administered intranasally to BALB/c mice together with an adjuvant (cholera toxin B subunit [CTB] supplemented with a trace amount of the whole toxin, CTB*) and its ability to induce innate immunity and confer protection against influenza was examined. Nasal wash virus titres 3 days after inoculation of homologous viruses were measured as an index of the ability of the vaccine to confer protection in mice immunized with either CTB*-combined vaccine or CTB* alone 1-21 days previously. The results were as follows. (1) Partial but significant reduction of the nasal-wash virus titres (prevention) was detected beginning 3 days after the vaccination, that is, 2 days earlier than the appearance of both virus-specific antibody-forming cells (AFCs) in the nasal-associated lymphoid tissue (NALT) and virus-specific IgA antibody responses in the nasal washes of mice immunized with the CTB*-combined vaccine. (2) The protection, detected on day 3 and peaking on day 5 but lost by day 21, was also conferred in mice immunized with CTB* alone. (3) The non-specific prevention was detected at doses of more than 0.3 microg of CTB*/mouse. (4) The nonspecific protection beginning 3 days after the immunization involved the enhanced expression of cytokine mRNAs (IL-15 and IL-18), considered responsible for natural killer (NK) cell activation, by the non-T cell populations in the NALT. (5) Normal NALT cells, when cultured in vitro with CTB*, secreted IL-1beta within a few hours in culture. These results demonstrate that the CTB*-combined vaccine, when given intranasally into mice, can confer nonspecific protection against influenza beginning 3 days after the vaccination and that CTB* also possessed this ability to confer protection non-specifically and temporarily by inducing the secretion of IL-1beta, one of the most important cytokines that initiates both innate and adaptive immunity, and also NK cell activity.

Involvement of antigen-presenting cells in the enhancement of the in vitro antibody responses by cholera toxin B subunit.

The enhancing effect of cholera toxin B subunit (CTB) on primary antibody responses to keyhole limpet haemocyanin (KLH) and the cellular basis of the effect were investigated, using in vitro cultures of mouse spleen cells. CTB (1-100 ng/ml) enhanced anti-KLH IgM, IgG and IgA antibody responses in a dose-dependent manner, when added to the cultures with KLH. This immunoenhancement was antigen specific, but not due to either polyclonal activation of the spleen cells or antigenic cross-reactivity between CTB and KLH. CTB did not affect the kinetics of the anti-KLH antibody responses. Early (Days 0-1) addition of CTB to the cultures enhanced the anti-KLH antibody production, whereas late (Days 5-7) addition of CTB did not. Addition of CTB with KLH to splenic adherent cells (SAC) resulted in a dose-dependent enhancement of the anti-KLH antibody responses, when the SAC were reconstituted with unimmunized non-adherent cells. Moreover, CTB enhanced IL-1 secretion from SAC incubated with KLH. These results suggest that CTB enhances the primary anti-KLH antibody responses in vitro by acting on early events in the responses, and that antigen-presenting cells play a major role in the enhancement.

Superior cross-protective effect of nasal vaccination to subcutaneous inoculation with influenza hemagglutinin vaccine.

Intranasal (i.n.) vs. subcutaneous (s.c.) administration of influenza hemagglutinin (HA) vaccine was systematically compared in BALB/c mice. Mice were immunized with different vaccines, together with cholera toxin B subunit as an adjuvant, and 4 weeks later were challenged with either a small (2 microliters) or a large (20 microliters) volume of mouse-adapted A/Guizhou-X (H3N2) virus, each of which gave virgin mice either a nasal or a lung predominant infection. Both i.n. and s.c. inoculations of A/Guizhou-X vaccine conferred almost complete protection against both challenges, i.n. inoculation of A/Fukuoka (H3N2) or A/Sichuan (H3N2) vaccine conferred almost complete cross-protection against 2-microliters challenge and a partial cross-protection against 20-microliters challenge, whereas the s.c. inoculation conferred no cross-protection against 2-microliters challenge with a partial cross-protection against 20-microliters challenge. Moreover, i.n. immunization of PR8 (H1N1) vaccine gave a slight cross-protection against 2-microliters challenge, while the s.c. inoculation did not. The degree of protection was easily improved by i.n. inoculation of higher doses of vaccine, but not by the s.c. inoculation. In parallel with the protection, the i.n. vaccination produced a high level of cross-reacting IgA and IgG antibody to A/Guizhou-X HA in nasal and broncho-alveolar washes, while the s.c. vaccination produced the cross-reacting IgG antibody alone. Thus, i.n. inoculation with inactivated vaccines, which induces cross-reacting anti-HA IgA antibody as well as IgG antibody, is more effective than s.c. vaccination for providing cross-protection against drift viruses.

H-2-unrestricted adjuvant effect of cholera toxin B subunit on murine antibody responses to influenza virus haemagglutinin.

Cholera toxin B subunit (CTB) has been shown to augment the antibody responses to influenza virus haemagglutinin (HA) in BALB/c mice immunized with HA vaccine together with CTB. In this study, mouse strain differences in the adjuvant effect of CTB on anti-HA antibody responses were investigated along with those in the antibody responses to CTB or HA, using various inbred and H-2 congenic strains. The antibody responsiveness to CTB depended on the H-2 haplotype of the strain: strains with the H-2b haplotype were high responders, those with H-2a, H-2k and H-2s were low responders, and those with H-2d were intermediate. The responsiveness to HA was also related to the H-2 haplotype: H-2a and H-2k strains were high responders, H-2b and H-2s strains were low responders, and H-2d strains were intermediate. However, the degree of the adjuvant effect of CTB on anti-HA antibody responses was almost constant, regardless of the H-2 haplotype or other genetic backgrounds of the strain. The lack of genetic restriction of the adjuvant effect would be favourable for application of CTB-combined HA vaccine to humans, who are genetically diverse. Moreover, these results suggest that the immunogenicity and adjuvanticity of CTB differ essentially in their mechanisms.

Enhancement of protective antibody responses by cholera toxin B subunit inoculated intranasally with influenza vaccine.

Effects of the B subunit of cholera toxin (CTB) on the primary antibody responses to influenza virus A/PR/8/34 (PR-8) (H1N1) HA vaccine and on protection against viral challenge were investigated in Balb/c mice which were immunized intranasally with both the vaccine and CTB. The dose of CTB (greater than or equal to 1 microgram) inoculated with the vaccine (greater than or equal to 0.15 microgram) induced high responses of both antiviral IgA antibodies in the nasal wash and haemagglutinin-inhibiting (HI) antibody in the serum, enough to provide complete protection against viral challenge four weeks after immunization. High levels of antibody were maintained for more than 16 weeks after inoculation, affording complete protection during this interval. The inoculation of HA vaccine prepared from influenza viruses A/Yamagata/120/86 (H1N1) or A/Fukuoka/C29/85 (H3N2) together with CTB provided partial protection against PR-8 infection, with production of antiviral IgA antibodies which were cross-reactive to PR-8 antigens whereas immunization with CTB and HA vaccine prepared from a different type of influenza virus (B/Ibaraki/2/85) failed to protect against PR-8 infection. These results indicate that CTB can produce an augmented and persistent antibody response to PR-8 HA vaccine, which is cross-protective to other A-type virus infections. The mechanisms by which CTB enhances the protective antibody responses to the nasally inoculated vaccine were investigated. The ability of CTB to augment antibody responses was lost, either when CTB was inoculated via the intravenous or subcutaneous route, or when CTB was introduced into nasal site one day before or after the vaccine inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pertussis toxin (PT) on T-cell populations sensitized for delayed-type hypersensitivity in mice.

Effects of pertussis toxin (PT) on sensitized T-cell populations for delayed-type hypersensitivity (DTH) were examined in mice. DTH was induced by sensitizing mice with ovalbumin (OA) and elicited by injecting OA into the footpad. DTH could be conferred on naive recipient mice by injecting sensitized spleen cells either intravenously into mice or locally into the footpad. When the sensitized mice were given PT at the time of DTH-elicitation, they did not express a high DTH reaction, with the maximum reaction 24 hr after elicitation. When the recipient mice were given PT just before intravenous injection of sensitized spleen cells, DTH was not conferred. In addition, when the sensitized spleen cells were treated with PT in vitro and then transferred intravenously, DTH was not conferred in recipient mice. However, DTH was conferred by local transfer of the sensitized spleen cells even after treatment with PT in vitro. Migration experiments using 51Cr-labeled, sensitized splenic T cells demonstrated that PT treatment of the T-cell population inhibited its accumulation in the DTH reaction site 24 hr after intravenous transfer. On the other hand, experiments on in vitro lymphokine production by the sensitized splenic T cells demonstrated that the PT treatment did not inhibit antigen-dependent production of a macrophage activating factor (MAF). These results suggest that PT suppresses the migration of the sensitized T-cell population from the circulation of the DTH reaction site but not their MAF production. Based on these findings, possible mechanisms by which PT affects DTH are discussed.

Properties of cloned T cells that mediate delayed-type hypersensitivity against ovalbumin in mice.

Cloned T-cell lines that mediate delayed-type hypersensitivity (DTH) against soluble protein antigen, ovalbumin (OA), were established in (C57BL/6 X DBA/2)F1 mice and their properties were examined. They induced antigen-specific delayed-type footpad reactions, characterized histologically by a predominant mononuclear cell infiltration, when transferred intravenously into syngeneic mice. Morphologically, they were medium or large lymphoblasts with granules in the cytoplasm and expressed Lyt 1 cell surface antigens. One of them proliferated antigen specifically under the presence of both C57BL/6 and F1 accessory cells, while others proliferated antigen specifically only under the presence of F1 accessory cells. They also produced macrophage-activating factor (MAF) and substances which mediate a DTH-like footpad inflammatory reaction with a maximum 6 hr after injection into the footpad of normal mice, when incubated in the presence of specific antigen and specific accessory cells in a serum-free medium for 24 hr. These results demonstrate that cloned DTH-effector T cells, established here against soluble protein antigen, are Lyt 1-positive, large lymphoblasts and that they produce MAF and footpad-reactive inflammatory substances antigen specifically under the presence of specific accessory cells.

Regulatory mechanism of delayed-type hypersensitivity in mice. IV. Effect of suppressor T cells on the development of memory T cells involved in accelerated generation of DTH-effector cells in vitro.

The effect of suppressor T cells (Ts) on the induction and the subsequent development of memory T cells for delayed-type hypersensitivity (DTH) was examined. The memory cells were induced in the spleens of mice primed previously with a low dose of reduced and alkylated ovalbumin (Ra-OA), and they generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion when cultured with OA in vitro. Ts were obtained from the spleens of mice which received OA-coupled spleen cells i.v. 4 days previously, and they inhibited antigen-specifically the induction of DTH responses in the recipient mice sensitized with alum-absorbed OA only when transferred with 5 weeks before sensitization. The spleen cells from mice given Ts together with the priming antigen 7 weeks before culture failed to generate DTH-Te in an accelerated manner on restimulation with OA in vitro. The memory cells from primed mice also did not cause accelerated generation of DTH-Te, when cultured with Ts in the presence of OA in vitro. These results indicate that both the induction of the memory cells by priming with antigen in vivo and the subsequent development of memory cells to DTH-Te by restimulation in vitro are inhibited independently by Ts. This corresponded well with the effect of Ts on the development of DTH-memory in vivo.

Regulatory mechanism of delayed-type hypersensitivity in mice. V. Augmentor cells for DTH responses.

Mice primed with 1 microgram of reduced and alkylated ovalbumin (RA-OA) developed not only long-lived memory cells for delayed-type hypersensitivity (DTH), capable of differentiating into DTH-effector T cells (DTH-Te) against ovalbumin (OA) when restimulated in vitro with OA, but also spleen cells capable of augmenting recipients' DTH responses to OA when transferred into cyclophosphamide (CY)-pretreated mice. The augmenting activity in spleen cells, upon transfer, was found 7 days, but not 21 or 91 days, after priming with RA-OA, although memory DTH-Te were present throughout the period of observation. The loss of augmenting activity after day 7 of priming was not due to the presence of suppressor cells; spleen cells taken 21 days after priming failed to suppress, upon transfer, the augmenting activity in 7-day-primed spleen cells as well as induction and expression of DTH responses to OA. When 7-day-primed spleen cells were fractionated on a discontinuous bovine serum albumin density gradient, the augmenting activity was found only in the medium-density-cell layer, although memory DTH-Te were separated in the high-density layer. Augmentation of DTH-Te generation could also be demonstrated in vitro when 7-day-primed spleen cells, but not 21-day-primed spleen cells, were added to cultures of spleen cells from CY-pretreated mice. These results indicate that, in the 7-day-primed spleen, there is an augmentor cell population which is different from memory DTH-Te and interacts with CY-resistant unprimed cells to facilitate DTH-Te generation.

Acute effects of cadmium on delayed-type hypersensitivity in mice.

A single exposure of mice to cadmium resulted in suppression of the induction of primary delayed-type hypersensitivity (DTH) responses as well as memory T-cell and suppressor T-cell activities, augmenting and inhibiting DTH respectively. Furthermore, cadmium suppressed the expression of already established DTH, in immune mice, even though DTH-effector T cells in the spleen of immune mice were not affected by cadmium injection. Such suppressive effects were demonstrated when cadmium was administered within 2 days before immunization or elicitation for DTH. Cadmium caused also within 2 days in mice thymic involution and splenomegaly. These results indicate that cadmium inhibits not only the generation of certain populations of T lymphocytes for DTH but also some mechanisms in the host involved in the expression of DTH.

Regulatory mechanism of delayed-type hypersensitivity in mice. III. In vitro analysis of memory T cells involved in augmentation of DTH responses.

The memory of delayed-type hypersensitivity (DTH), manifested by the augmented responsiveness upon challenge with alum-absorbed ovalbumin (OA), was induced in mice primed 7 days, 21 days, or 90 days previously with 1 microgram of reduced and alkylated OA. The memory cells involved in the augmentation of DTH responses were analyzed in the in vitro induction system of T cells which mediate DTH against OA. Spleen cells from the primed mice generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion, compared with unprimed spleen cells, when cultured with OA. The accelerated generation of DTH-Te in vitro was induced antigen specifically and was dependent on a certain T cell population in the primed spleen. The T cell population was found in the spleen of primed mice for at least 3 months after priming, corresponding to the persistence of DTH-memory in vivo. Moreover, it was fractionated in the high-density layer by discontinuous bovine serum albumin gradient centrifugation. The high-density cell population decreased in density with increase in the time of culture and developed into DTH-Te, which were separated in the low-density layer on day 4 of culture. These results indicate that the T cells involved in the accelerated generation of DTH-Te in vitro are long-lived DTH-memory T cells, which are probably precursor cells, capable of differentiating into DTH-Te upon challenge with the antigen.

Correlation between production of inflammatory substances and generation of effector T cells mediating delayed-type hypersensitivity in mice.

In vitro exposure to human serum albumin (HSA) of splenic lymphocytes from mice sensitized for delayed-type hypersensitivity (DTH) against HSA resulted in the release of substances that could induce a footpad inflammatory reaction with a maximum of 6 hr after injection into normal mice. The substances were fractionated mainly in a molecular weight range of 30,000 to 70,000 daltons on Sephadex G-200. The ability of sensitized lymphocytes to produce the substances was dependent on T cells, was antigen specific, and correlated well with the ability of the lymphocytes to mediate DTH reactions. Moreover, the substances were produced efficiently by the DTH effector cell population generated in the in vitro culture system and also by the effector cell-enriched fractions on discontinuous bovine serum albumin gradients. These results suggest that the substances are produced by DTH-effector cells.

Preferential induction of memory T cells for delayed-type hypersensitivity with reduced and alkylated human serum albumin in mice.

Reduction and alkylation of human serum albumin (HSA) resulted in molecular aggregation of the protein. The reduced and alkylated antigen (RA-HSA) was lacking in the ability to induce delayed-type hypersensitivity (DTH) response as well as antibody response to native HSA in mice, although native HSA induced both responses. On the other hand, RA-HSA could stimulate a priming function that accelerated and enhanced the DTH response to native HSA, which, however, failed to stimulate the function. Thus, DTH-related memory activity was dissociated from DTH-related effector activity. The DTH-related memory activity, manifested by the accelerated an enhanced response in RA-HSA-primed mice, could be transferred antigen-specifically by their spleen cells and T-cell enriched fraction, but not by their T-cell depleted spleen cells. The RA-HSA-primed T cells, however, failed to transfer the effector function for DTH response. These results suggested that DTH-related memory T cells belong to different subset(s) of T cells from effector T cells for a DTH response.

Regulatory role of suppressor T cells in the expression of delayed-type hypersensitivity in mice. II. Soluble factor from thymic suppressor cells stimulated with antigen in vitro and its possible interaction with macrophages.

Thymus cells from mice primed s.c. with a high dose (10 mg) of lysozyme (Lys) specifically suppressed delayed footpad reaction (FPR) in mice previously immuned with lipid-conjugated lysozyme (D.Lys), and also suppressed the transfer of FPR by D.Lys-immune spleen cells into normal mice. Furthermore, they inhibited antigen-stimulated DNA synthesis of D.Lys-immune spleen cells in vitro. If the suppressor thymus cells were cultured with Lys in vitro, they produced soluble factor which depressed the ability of D.Lys-immune spleen cells to transfer FPR. Both supernatant of culture without Lys and extract of suppressor thymus cells were inactive in supression of FPR. The suppressor factor was antigen-specific because its suppressive activity was absorbed with Lys but not with an unrelated antigen lactalbumin. The factor failed to depress the ability of D.Lys-immune spleen cells to transfer FPR when the spleen cells were depleted of glass-adherent cells. In addition, incubation of peritoneal exudate cells from normal mice with the factor rendered the cells suppressive for passive transfer of FPR. These results suggest that the suppressor factor depresses the effector function of T cells responsible for FPR possibly via macrophage.

Reaginic antibody formation in the mouse. X. Possible role of suppressor T cells in transient IgE antibody responses.

The kinetics of IgE antibody response to alum-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) was dependent on the dose of immunogen. A persistent IgE antibody response was obtained when high responder BDF1 mice were immunized with a minimum (0.05 microgram) dose. An increase of the immunogen to 10 microgram depressed IgE antibody responses but enhanced IgG antibody responses of both hapten and carrier specificities. Determination of T helper cell activity and B memory cells after immunization with different doses of antigen indicated that minimum immunogen was favorable for developing helper activity, whereas 1 to 10 microgram immunogen were more favorable than a 0.05-microgram dose for developing both IgE and IgG B memory cells. Nevertheless, neither helper T cells nor B memory cells in the spleen explains a transient IgE antibody response to a high (10 microgram) dose of DNP-OA. Evidence was obtained that immunization with 10 microgram OA induced generation of antigen-specific suppressor T cells, which were not detectable after immunization with 0.05 microgram OA. Transfer of suppressor T cells to DNP-OA-primed mice depressed both anti-hapten and anti-carrier IgE antibody responses. The results suggested strongly that suppressor T cells are involved in a transient IgE antibody response to a high-dose immunogen.

Cellular and humorial immune responses in mice. III. Acceleration of delayed hypersensitivity response by presensitization with suboptimal dose of antigen.

Delayed hypersensitivity (DH) response in mice induced by subcutaneous (s.c.) injection of optimal dose of sheep red blood cells (SRBC) 10(8)) was accelerated by s.c. injection of the antigen of 10(3) or more doses, given 2 or more days earlier. The accelerated response appeared soon after the injection of optimal antigen dose, that is, 1 or 2 days earlier than the response of non-presensitized control. The acceleration was antigen specific. The accelerated response was generally accompanied by an acceleration and/or enhancement of humoral antibody response. Parallel to the acceleration of DH response, the proliferation of regional lymph node cells in the presensitized mice was induced immediately after the following injection of 10(8) SRBC, 1 day earlier than that of non-presensitized animals. These results suggest that presensitization of mice with the antigen induces DH-related memory cells which proliferate immediately after the following injection and function as effector cells for DH reactions, and that the development of DH-related memory cells occurs in close relation to that of helper thymus-derived (T) cells for antibody production.

Cellular and humoral immune responses in mice. II. Effect of intraperitoneal or subcutaneous injection of carrier of anti-hapten antibody and delayed hypersensitivity responses.

The subcutaneous (s.c.) injection of sheep red blood cells (SRBC) without adjuvant into mice preferentially induced delayed hypersensitivity (DH) reaction, as measured by footpad swelling, while intraperitoneal (i.p.) measured by the haemagglutinin test. Under these conditions, the properties of the helper activities of thymus-derived (T) cells for humoral responses were examined, in association with the features of the DH response, by measuring the anti-hapten and anticarrier antibody responses after a booster injection of trinitrophenylated (TNP) SRBC and by changing the combination of doses and injections routes of the carrier and the hapten-carrier conjugates. When mice were presensitized with i.p. injections of SRBC and boosted with i.p. injections of TNP-SRBC, the anti-TNP antibody production was maximally enhanced by presensitization with a low dose of SRBC, and gradually abolished with higher doses of SRBC for pre-sensitization. In the latter case, anti-SRBC antibody production was increases with increasing doses of SRBC..