Use of prior vaccinations for the development of new vaccines.

There is currently a need for vaccine development to improve the immunogenicity of protective epitopes, which themselves are often poorly immunogenic. Although the immunogenicity of these epitopes can be enhanced by linking them to highly immunogenic carriers, such carriers derived from current vaccines have not proven to be generally effective. One reason may be related to epitope-specific suppression, in which prior vaccination with a protein can inhibit the antibody response to new epitopes linked to the protein. To circumvent such inhibition, a peptide from tetanus toxoid was identified that, when linked to a B cell epitope and injected into tetanus toxoid-primed recipients, retained sequences for carrier but not suppressor function. The antibody response to the B cell epitope was enhanced. This may be a general method for taking advantage of previous vaccinations in the development of new vaccines.

A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1.

The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli. Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P. falciparum blood stage parasite challenge. The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code). To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl). The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E. coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient. Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites. The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite.

Maturation of the lymphoid system. IV. Ontogenetic compartmentalization of T cell function.

Pretreatment of one day old but not eight day old or adult A/J mice with soluble ovalbumin (OVA) initiated specific T-cell unresponsiveness as reflected both in T-cell dependent cellular proliferation and in anti-hapten antibody responses to dinitrophenyl-OVA. In contrast, injection of soluble human gamma globulin into either neonatal or adult A/J mice resulted in unresponsiveness. The ability of lymph node T-cells to be sensitized by protein antigens occurred shortly after birth, since the degree of sensitization in 9 and 26 day old mice was similar. Finally, a striking ontogenetic difference was noted in the capacity of lymphocytes derived from the lymph nodes and spleens of young mice to respond to T-cell mitogens. Thus, while splenocytes obtained from 9 day old mice exhibited meager responses to PHA and Con A, lymph node cells from these animals responded at nearly adult levels. These observations are interpreted as reflecting an ontogenetic and tissue-specific division of T-cell function.

Maturation of the lymphoid system. III. Induction of selective unresponsiveness by injection of a haptenated carbohydrate into neonatal mice.

Neonatal treatment of A/J mice with DNP-Ficoll reduced or eliminated indirect anti-DNP PFC normally produced in response to adult challenge with DNP-keyhole limpet hemocyanin. The remaining direct anti-DNP PFC response was of low avidity. Spleen cells from neonatal A/J mice inhibited the in vitro but not the in vivo response of adult spleen cells to DNP-Ficoll.

[Vaccination against malaria: initial trial with an ant-sporozoite vaccine, (NANP)3-TT (RO 40-2361) in Africa (Bobo-Dioulasso, Burkina Faso)]. / Vaccination contre le paludisme: premier essai avec un vaccin antisporozoïte, le (NANP)3-TT (RO 40-2361) en Afrique (Bobo-Dioulasso, Burkina Faso).

The vaccine (NANP)3-TT is a synthetic peptide of the circumsporozoite protein (CS) of Plasmodium falciparum coupled to tetanus toxoid (TT) as protein carrier and adsorbed to aluminium hydroxide as adjuvant. The objectives of the study were to assess the immunogenicity and the protective efficacy of the vaccine in an area where malaria is endemic. The study was conducted in a zone of irrigated rice cultivation known as the Vallée du Kou to the North of Bobo-Dioulasso. Malaria transmission is permanent in the Vallée with maxima in July and November. The study was conducted from June to December 1988. It was a controlled randomised, double blind, prospective vaccine trial. A total of 123 infants from 3 to 5 months of age were randomly assigned to three groups. Group I (controls) received three doses of TT alone, group II received two doses of TT and one of (NANP)3-TT and group III received three doses of (NANP)3-TT. These vaccines were administered simultaneously with the Enlarged Program of Immunisation (EPI) vaccines. The clinical parasitological and immunological status of the children was then monitored over a period of five months. No systemic reactions to the vaccine were observed in the infants either immediately after administration or during the follow-up. Minor local tumefactions were observed in only 3% of the children. The vaccine was found to be immunogenic with a peak IgG response at day 75, when 56% (group II) and 60% (group III) showed antibody titres of at least four times that seen at day 0. The response, however, was a short duration; by day 150 the average antibody titres were not significantly different between the three groups. The incidence and the level of parasiaemia and the incidence of clinical malaria were also not significantly different for each of the three groups during the period of the study. The association of (NANP)3 with tetanus toxoid was not shown to be immunologically inhibitive. The results, despite not showing a protective effect for the vaccine (NANP)3-TT, have shown its immunogenicity and therefore suggest that further development of this vaccine may be worthwhile.

Carrier sequence selection--one key to successful vaccines.

The trend towards epitopic vaccines has brought with it the problem of ensuring carrier function, a role previously filled by carrier sequences of the attenuated organism. Here, Howard Etlinger proposes the use of carrier epitopes, derived from vaccines already in use and selected for their ability to activate only helper T-cell responses, in the administration of B-cell-specific epitopic vaccines.

Is the antibody response specific?

Introduction of heterologous immunoglobulin (Ig) or red blood cells into mice results in an increased production of Ig and immunogen-reactive antibody. Since the increase in both total Ig and specific antibody is similar, it is concluded that the antibody response is specific. This result, which contrasts with those of previous studies in which the production of large amounts of nonspecific Ig was reported, suggests that in vivo T-dependent B cell activation and differentiation to plasma cells requires the presence of immunogen and linked recognition.

Overcoming inhibition of antibody responses to a malaria recombinant vaccinia virus caused by prior exposure to wild type virus.

Pre-injection of mice with vaccinia virus inhibited the subsequent antibody response to a recombinant polypeptide expressed by vaccinia virus. The inhibition was overcome following additional challenges with recombinant vaccinia virus. This suggests that a potential disadvantage in vaccinia-immune individuals can be circumvented and may be outweighed by the advantages of the vector.

Model using a peptide with carrier function for vaccination against different pathogens.

It has been shown that pre-immunization with a protein can inhibit the antibody response to a new B-cell sequence which is coupled to the protein (epitope-specific suppression). By utilizing a peptide with carrier function from the protein, rather than the entire protein, the antibody response to the new B-cell sequence is enhanced in protein-primed mice. The present results extend this observation by showing that mice which have been pre-immunized with a protein followed by a B-cell sequence linked to a carrier peptide from the protein produce an enhanced antibody response to a new B-cell sequence linked to the same carrier peptide sequence. This indicates that it would be possible to reuse a carrier peptide sequence coupled with newly defined protective B-cell epitopes in a given individual to achieve immunity against new pathogens.

T15 dominance in BALB/c mice is not controlled by environmental factors.

The effects of the environment on the expression of T15 in the in vivo anti-PC response of BALB/c mice were analyzed. T15 dominance in young BALB/c mice was independent of the expression of T15 dominance in either parent, because the offspring of parental mice that were suppressed for T15 production presented antibody responses dominated by the T15 idiotype. Also, dominant T15 expression was independent of living microorganisms; mice raised in conventional, specific pathogen-free or germfree conditions mounted similar T15 dominant antibody responses. Furthermore, T15 expression was independent of the conventional diet, because mice raised on a synthetic diet produced T15-dominant antibody responses. Moreover, mice that received a synthetic diet under germfree conditions also produced T15 dominant antibody responses. Thus, the generation of T15 dominance in BALB/c mice appears to be independent of environmental factors and within the context of the present and earlier results, originates at the level of B cell-mediated clonal selection/regulation, genetic mechanisms concerning Ig gene rearrangement and expression and/or the fine specificity of the combining site for antigen on the B cell.

Complement activation by female protein, the hamster homologue of human C-reactive protein.

The capacity of Syrian hamster female protein (FP), a phosphorylcholine (PC)-binding pentraxin, to activate complement was tested in an in vitro system consisting of PC-coupled sheep red blood cells and guinea pig serum as the complement (C) source. FP was demonstrated to fix complement as reflected by hemolysis. Such hemolysis was eliminated by heat treatment (56 degrees C, 30 min) of guinea pig serum and inhibited by PC chloride but not dinitrophenyl lysine. The inability of C4-deficient guinea pig serum to provide lytic activity indicated that lysis proceeded through the classical hemolytic pathway.

Netwhat?

The hallmark of an antibody response is considered to be its specificity for the immunogen. Nonetheless, most antibody elicited by an immunogen has been reported to be unreactive with it. We have evaluated specificity by characterizing the primary antibody response of mice to heat-aggregated human gamma-globulin with four approaches: (a) quantification of antibody-producing cells; (b) hybridoma analysis; (c) in situ antigen binding and (d) analysis of secreted antibody. The results show that the nonspecific component of this response is negligible. These observations suggest that such a component is not a basic feature of the antibody response and it is discussed that nonantigen-specific antibody may arise from a variety of causes many of which are artifactual.

Towards a synthetic malaria vaccine: cyclization of a peptide eliminates the production of parasite-unreactive antibody.

In a previous study, human beings were vaccinated with a P. falciparum malaria vaccine candidate consisting of tetanus toxoid coupled to linear (Asn-Ala-Asn-Pro)3 ((NANP)3). The vaccine initiated protection in some people, but some individuals mainly produced anti-peptide antibodies that did not react with the pathogen. A likely contributor to the formation of epitopes that give rise to pathogen-unreactive antibodies is the free terminal proline which is not a terminal residue in the native protein. To avoid the elicitation of antibodies against terminal epitopes, (NANP)3 was cyclized. In contrast to monoclonal antibodies to the linear peptide where 35% were unreactive with the parasite, all monoclonal antibodies to the cyclized peptide were found to react with the parasite.

Lack of a requirement for idiotype matching: T cells from mice which cannot produce idiotype support idiotype-positive antibody response.

A requirement for idiotype matching was reported such that carrier-primed T helper cells from mice, which are unable to produce an idiotype (idiotype-negative) because of genetic reasons or adult anti-idiotype or neonatal anti-mu antibody treatment, did not provide helper function for an idiotype-positive antibody response. We have analyzed the requirement for idiotype matching in the response to phosphorylcholine by using mice which are idiotype-negative because they were injected with anti-idiotype antibody shortly after birth. Carrier-primed T cells from such animals supported idiotype-positive responses when mixed with normal or primed B cell populations and challenged with appropriate antigen; these responses were quantitatively and qualitatively similar to those obtained with T cells from idiotype-positive animals. These results demonstrate no requirement for idiotype matching and suggest that the procedure used to establish an idiotype-negative T cell donor may be decisive in showing a requirement for idiotype matching.

Alteration of immunologic function through early ontogenetic experiences. Selective inactivation of T15-positive B cells in neonatal mice is related to receptor avidity and is independent of thymus function.

The immunologic effects initiated by injecting neonatal mice with phosphorylcholine (PC)-protein conjugates were analyzed. Pretreated nude or thymus-bearing mice produced greater proportions of low-avidity, TEPC 15-negative antibody than control animals when challenged as adults with thymus-dependent (TD) or -independent (TI) PC-conjugated antigen. Furthermore, pretreatment had a greater inhibitory effect on responses elicited by TD compared with TI antigen. These results demonstrate that exposure of neonatal mice to PC-protein conjugates results in the selective inactivation of higher avidity, T15-positive, PC-reactive B cells; this process is independent of thymic function and the duration of hyporesponsiveness is linked to the relative T dependency of the challenge antigen.

Maturation of the lymphoid system. I. Induction of tolerance in neonates with a T-dependent antigen that is an obligate immunogen in adults.

A/J mice displayed a striking ontogenetic difference in the capacity to respond to DNP-Ficoll, a T-independent antigen, and to aggregated human gamma-globulin (AHGG), a T-dependent antigen. Thus, whereas responses to DNP-Ficoll of 4-day-old mice were similar in magnitude to those of adult animals, responses to AHGG did not become pronounced until mice were some 30 to 40 days of age. The inability of young animals to respond to AHGG was reflective of a negative consequence of lymphocyte/antigen interaction, since such mice became specifically unresponsive to subsequent challenges with AHGG. Unresponsiveness induced by neonatal injection of AHGG lasted 50 to 60 days, in contrast to that induced by deaggregated HGG, which persisted some 100 days longer. The unresponsive state induced by injection of neonates with AHGG maintained itself upon adoptive transfer and did not appear to be linked to suppressive factors associated with either serum or lymphoid cells for its maintenance. Finally, AHGG was also shown to be capable of inducing unresponsiveness in neonatal, athymic mice. These results demonstrate that AHGG, the normally immunogenic form of HGG in adult mice, can serve as an effective tolerogen when administered into a neonatal environment.

Maturation of the lymphoid system. II. Characterization of the cellular levels of unresponsiveness induced in neonates by a T-dependent antigen that is an obligate immunogen in adults.

It has previously shown that AHGG, a form of HGG that is highly immunogenic in euthymic adult mice, is capable of inducing specific unresponsiveness when injected into neonatal animals. This report extends this finding and indicates that such a neonatal treatment results in the induction of tolerance in T as well as B cells. Furthermore, a similar conclusion was reached regarding specific T lymphocyte function in animals treated as neonates with OVA. The ability of LPS to modulate responses of neonatal animals to AHGG or DHGG was also examined. It appeared that such mice were not susceptible to the adjuvant effects of LPS until the 4th week of life. Furthermore, LPS was incapable of inhibiting the unresponsiveness induced in mice by either DHGG or AHGG until the 3rd or 4th week of life.

Expression of a distinct B cell clonotype profile after recovery from antigen-induced unresponsiveness.

The purpose of these experiments was to determine whether the B cell clonotype profile expressed in mice which have recovered from antigen-induced unresponsiveness is similar to that of nontolerized mice. Unresponsiveness to phosphorylcholine (PC) was initiated by injection of neonatal mice with PC-coupled human gamma globulin, resulting in the inability to respond to challenge with PC-lipopolysaccharide at 1.5 months of age. By 3 months of age, pretreated mice were 50% responsive while by 5 and 7.5 months of age, full responsiveness was observed. At each time point two differences distinguished the anti-PC antibody from nonpretreated mice. First, whereas nonpretreated mice displayed T15 dominance, pretreated mice did not. Second, the average avidity of T15-negative antibody produced in pretreated mice was greater than that in nonpretreated mice and similar to that of T15-positive antibody. Possible mechanisms for this "permanent" alteration of the antibody profile are discussed.