Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.
Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-1 6101 env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant envspecific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level revindependent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.
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