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Smoking remains one of the major causes of morbidity and mortality worldwide. One approach to assisting smoking cessation is via anti-nicotine vaccines, composed of nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). We have previously shown that the carrier, hapten, linker, hapten load, degree of conjugate aggregation, and presence of adducts can each influence the function (nicotine-binding capacity) of the antibody (Ab) induced. Herein, we extend those findings and show that tertiary structure is also critical to the induction of functional immune responses and that this can be influenced by conjugation conditions. We evaluated immunogenicity in mice using six lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7), and a single point (glycine 52 to glutamic acid) mutant nontoxic form of diphtheria toxin, cross-reactive material 197 (CRM197), which were synthesized under different reaction conditions resulting in conjugates with equivalent molecular characteristics (hapten load, aggregates, adducts), but a different tertiary structure. When tested in mice, better functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with conjugates with a more closed structure than those with an open conformation. These studies highlight the need for a better understanding of the physicochemical properties of small molecule conjugate vaccines.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

Treatment with an oligodeoxynucleotide (ODN) containing CPG motifs (CpG ODN 7909) was found to protect BALB/c mice from lung infection or death after aerosol challenge withBurkholderia mallei. Protection was associated with enhanced levels of gamma interferon (IFN-γ)-inducible protein 10, interleukin-12 (IL-12), IFN-γ, and IL-6. Preexposure therapy with CpG ODNs may protect victims of a biological attack from glanders.

Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7–CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7–CRM.

Tobacco smoking is one of the most preventable causes of morbidity and mortality, but current smoking cessation treatments have relatively poor long term efficacy. Anti-nicotine vaccines offer a novel mechanism of action whereby anti-nicotine antibodies (Ab) in circulation prevent nicotine from entering the brain, thus avoiding the reward mechanisms that underpin nicotine addiction. Since antibody responses are typically long lasting, such vaccines could potentially lead to better long-term smoking cessation outcomes. Clinical trials of anti-nicotine vaccines to date have not succeeded, although there was evidence that very high anti-nicotine Ab titers could lead to improved smoking cessation outcomes, suggesting that achieving higher titers in more subjects might result in better efficacy overall. In this study, we evaluated CpG (TLR9 agonist) and aluminum hydroxide (Al(OH)3) adjuvants with a model anti-nicotine antigen comprising trans-3'aminomethylnicotine (3'AmNic) conjugated to diphtheria toxoid (DT). Anti-nicotine Ab titers were significantly higher in both mice and non-human primates (NHP) when 3'AmNic-DT was administered with CpG/Al(OH)3 than with Al(OH)3 alone, and affinity was enhanced in mice. CpG also improved functional responses, as measured by nicotine brain levels in mice after intravenous administration of radiolabeled nicotine (30% versus 3% without CpG), or by nicotine binding capacity of NHP antisera (15-fold higher with CpG). Further improvement should focus on maximizing Ab function, which takes into account both titer and avidity, and this may require improved conjugate design in addition to adjuvants.

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8(+) T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8(+) T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8(+) T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4(+) T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8(+) response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.

Etude experimentale, par la methode de suspension de fines particules d'aluminium de la microstructure de l'ecoulement a l'interieur de gouttelettes en cours d'evaporation sur une plaque chaude plane. Etude effectuee avec des lignes instables, substables et stables