Vaccination to protect against human infectious diseases may be enhanced by using adjuvants that can selectively stimulate immunoregulatory responses. In a murine model, a novel nanoparticulate adjuvant composed of calcium phosphate (CAP) was compared with the commonly used aluminum (alum) adjuvants for its ability to induce immunity to herpes simplex virus type 2 (HSV-2) and Epstein-Barr virus (EBV) infections. Results indicated that CAP was more potent as an adjuvant than alum, elicited little or no inflammation at the site of administration, induced high titers of immunoglobulin G2a (IgG2a) antibody and neutralizing antibody, and facilitated a high percentage of protection against HSV-2 infection. Additional benefits of CAP include (i) an insignificant IgE response, which is an important advantage over injection of alum compounds, and (ii) the fact that CAP is a natural constituent of the human body. Thus, CAP is very well tolerated and absorbed. These studies were performed with animal models. By virtue of the potency of this CAP adjuvant and the relative absence of side effects, we believe that this new CAP formulation has great potential for use as an adjuvant in humans.Historically, adjuvants have been necessary to improve vaccine efficacy in order to afford protection against infections. A key reason for this is that both attenuated virus preparations and, particularly, recombinant proteins are often poorly antigenic. In the past decade, several adjuvants have been evaluated in clinical trials. Calcium phosphate (CAP), MF59, aluminum (alum) compounds, and virosomes have been approved for human use in several European countries (23). In the United States, alum compounds are the most extensively used adjuvants in licensed vaccines for humans. Although they effectively enhance immune responses, there are several disadvantages associated with their use (3,5,14). The disadvantages of alum-based adjuvants include the severity of local tissue irritation, the longer duration of the inflammatory reaction at the injection site, strong Th2 responses, minimal induction of cell-mediated immunity, and a propensity to elicit undesirable immunoglobulin E (IgE) responses (11,12,17,27). Alum compounds have also been shown to increase the levels of potential undesirable homocytotropic antibodies in animal species (9, 21). Furthermore, alum-based vaccines are frequently ineffective for the induction of antiviral immunity (4). For these reasons, new adjuvants are being developed to enhance the immunity against weak antigens. New-generation adjuvants are designed to induce minimal side effects, enhance the duration of the immune response, and concurrently stimulate humoral, cellular, and mucosal immune responses. Furthermore, an ideal adjuvant would be biodegradable, economical, and simple to manufacture. In addition, it would have the potential to selectively trigger a defined class of immune response such as the T-helper 1 (Th1) CD4 ϩ T-cell response and cell-mediated immunity and have equal applicability for any new-genera...

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2؉CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2؉CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2؉CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2؉CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.Since mucosal surfaces act as the primary point of entry for most pathogens and the first line of defense against them, vaccines inducing effective mucosal immunity may reduce rates of infection and decrease the morbidity and mortality of infectious diseases. Currently, no safe and effective mucosal vaccine adjuvants are approved for human use.Mucosal vaccine delivery is a promising strategy. Mucosal vaccines administered in one part of the body can elicit an antibody response in mucosal tissues remote from the site of initial antigen exposure. This effect occurs because of the common mucosal immune system (13). A major obstacle to developing a mucosal vaccine in humans is finding a safe and effective adjuvant. Experimental mucosal adjuvants include cholera toxin, heat-labile enterotoxin, mutant toxins (LTK63 and LTR72), CpG oligodeoxynucleotide, polymerized liposomes, microparticles, and interleukins or immune modulators. None of these adjuvants is approved for use in humans (3,12,18).Biodegradable calcium phosphate particles have been investigated as an alternative to aluminum adjuvants for parenteral vaccines. Clinical studies conducted in France described the use of a calcium phosphate adjuvant for secondary or booster immunizations against diphtheria and tetanus (8). Calcium phosphate has also been used for allergen desensitization (6, 16). Early studies indicated that calcium phosphate particles produce strong adjuvant effects, induced less immunoglobulin E (IgE) than aluminum adjuvants, and elicited only minimal local irritation in animal experiments and human clinical trials (6,8,11,17).Here, we describe a unique formulation of calcium phosphate nanoparticles (CAP) which is distinct from the formulations of calciu...

KEYWORDS:pulmonary delivery, insulin, CAP-PEG particles, pharmacokinetics, pharmacodynamicsThe purpose of the study was to evaluate the influence of calcium phosphate (CAP) and polyethylene glycol (PEG) particles on the systemic delivery of insulin administered by the pulmonary route. Two methods of pulmonary delivery were employed: intratracheal instillation and spray instillation. Insulin-CAP-PEG particles in suspension (1.2 U/kg, 110-140 μL) were administered to the lungs of fasted rats by intratracheal instillation (INCAPEG) or spray instillation (SINCAPEG). Control treatments consisted of insulin solution (1.2 U/kg) by intratracheal instillation, spray instillation, and subcutaneous administration (SC). Plasma concentrations of insulin and glucose were determined by chemiluminescence and colorimetric methods, respectively. Data were analyzed by compartmental and non-compartmental methods, and pharmacokinetic (PK) and pharmacodynamic (PD) parameters of insulin disposition were determined. PK analysis suggested that insulin administered in particles had a longer half-life, a longer mean residence time, and a smaller rate of elimination than insulin in solution. In addition, insulin bioavailability after SINCAPEG was 1.8-fold that of insulin solution administered SC. PD analysis showed that smaller areas under the effect curve and, conversely, larger areas above the effect curve were obtained after INCAPEG in comparison to insulin solution. The magnitude of this effect was increased after SINCAPEG. The presence of CAP-PEG particles appears to positively influence the disposition of insulin administered to the lungs of Sprague-Dawley rats. Spray instillation appears to be a more efficient method of delivering insulin to the lungs of rats than intratracheal instillation.

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.

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