Enhancement of HIV type 1 vaccine immunogenicity by block copolymer adjuvants. I. Induction of high-titer, long-lasting, cross-reactive antibodies of broad isotype.

Improvements in HIV-1 vaccines are urgently needed since many of the available vaccines are weak immunogens. We examined the ability of CRL1005, a novel nonionic block copolymer adjuvant, to improve the immunogenicity of multiple HIV-1 envelope vaccines: six gp120s and single and multiple V3 peptides (MAPs). Formulation of vaccine with adjuvant, as compared with alum or saline, enhanced antibody titer in mice up to 200-fold, with antibody half-lives of >200 days. For most vaccinations, an oil-in-water formulation induced the highest antibody titers; for some antigens, however, particularly single peptides, water-in-oil (w/o) was better. Antigen cross-reactivity was optimized by formulation in w/o, while addition of detoxified lipopolysaccharide enhanced levels of IgG2a and IgG2b. After more than 1 year of observation, no vaccine-related toxicity was observed and emulsified antigen in encapsulated depots was found at immunization sites of w/o-immunized animals. No other adjuvant has been reported to induce such long-lasting antibodies, and the ability of CRL1005 to greatly amplify and qualitatively modify antibody responses suggests that it may be useful in developing improved HIV vaccines for humans.

Induction of long-lasting immunity to Plasmodium yoelii malaria with whole blood-stage antigens and copolymer adjuvants.

We previously reported that protection of mice from nonlethal Plasmodium yoelii malaria by immunization with whole killed blood-stage parasites was dependent on the adjuvant and that adjuvants influenced both the specificity and isotype of Ab. Additional studies with the most effective formulations were undertaken to better define the protective responses and 100% protection from lethal P. yoelii malaria was produced by three immunizations with Ag in copolymer P1004 and detoxified RaLPS as adjuvants and 83% protection was induced by a single immunization. The protection lasted for 9 mo and was associated with an anamnestic rise in Ab titer of the IgG2a isotype during the challenge infection. Passive immunization with Ab from animals that had been immunized and challenged transferred sterile immunity. Splenectomy reduced, but did not abolish, protection. These data suggest that the effective Ab is directed against labile epitopes on the surface of blood-stage parasites. The vaccines primed animals for production of such Ab, but its synthesis was efficiently induced only by challenge with live organisms.

A comparison of commercially available adjuvants for use in research.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

A neutralizing monoclonal antibody against Coxsackievirus B4 cross-reacts with contractile muscle proteins.

A panel of Coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies (mAbs) were tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Immunohistochemical studies revealed an A band pattern of staining of the heart. Examination of sequential differential extracts of heart by Western immunoblotting showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. These studies imply that molecular mimicry is one mechanism by which autoimmunity could develop during CVB4 induced myocarditis.

Reactivation of affinity-purified estrogen receptors by peptides derived from histone H2B.

Purification of estrogen receptors by affinity chromatography over diethylstilbestrol-agarose is associated with a major loss of estradiol binding activity. Histone H2B can restore a significant fraction of the binding activity. Cleavage of the H2B molecule into two halves by cyanogen bromide reveals that the carboxyl terminus is responsible for the major reactivating effects.

Characterization of rabbit uterine estrogen receptor proteins by radioiodination and partial peptide mapping.

Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.

An immobilized antiestrogen binds a specific uterine protein in addition to estrogen receptor proteins.

An antiestrogen affinity resin was synthesized by conjugating LY117018, a benzothiophene -derived antiestrogen, to epoxy-activated agarose. This affinity resin bound the Mr = 50 000 and 65 000 estrogen receptor proteins of rabbit uterine cytosol; in addition, it retained a protein from the cytosols of both rat and rabbit uteri that exhibited an ability to interact specifically with LY117018. The possibility that the LY117018 binding protein, which is distinct from estrogen receptors, may play a role in the antiestrogenic actions of LY117018 is discussed.