Recommendations and best practices for reference standards and reagents used in bioanalytical method validation.

The continued globalization of pharmaceutics has increased the demand for companies to know and understand the regulations that exist across the globe. One hurdle facing pharmaceutical and biotechnology companies developing new drug candidates is interpreting the current regulatory guidance documents and industry publications associated with bioanalytical method validation (BMV) from each of the different agencies throughout the world. The objective of this commentary is to provide our opinions on the best practices for reference standards and key reagents, such as metabolites and internal standards used in the support of regulated bioanalysis based on a review of current regulatory guidance documents and industry white papers for BMV.

A minimum CR2 binding domain of C3d enhances immunity following vaccination.

The degradation product of the third (C3) complement component, C3d, links innate and adaptive immunity, and the covalent attachment of C3d to an antigen enhances antigen-specific immune responses. C3d has been hypothesized to enhance immunity by direct interaction with complement receptor 2 (CR2/CD21) on immune cells. However, the domains on C3d important for CR2 binding have been controversial, with various studies reaching contradictory conclusions. In addition, the concept of B-cell activation via CR2 by C3d has been questioned, since mice lacking CR2 still elicit C3d-enhanced immunity following vaccination. Therefore, the goal of this study was to determine if a peptide representing one of the proposed CR2 binding domains of C3d could substitute for the entire protein and enhance antigen-specific immunity. Mice (BALB/c) were vaccinated with the HIV-1 gp120 envelope glycoprotein (Env(gp120)) alone or fused to multiple copies of the murine C3d or a twenty-eight amino-acid peptide (P28) containing a minimum CR2 binding domain. Each immunogen was expressed from DNA plasmid in vivo or injected as purified recombinant protein. The fusion of the P28 peptide to Env(gp120) enhanced both humoral and cell-mediated immune responses with similar efficiency as Env(gp120) conjugated to C3d. The fusion of C3d or P28 to Env(gp120) elicited higher-titer anti-Env specific antibody, enhanced avidity maturation of the elicited antibody, and elicited higher numbers of IFN-gamma and IL-4 secreting cells compared to Env(gp120) immunizations. This CR2-binding domain specific 28 amino acid peptide can substitute for the entire C3d molecule and enhance immunity. These results indicate that the adjuvant properties of C3d are associated with CR2 interaction.

HIV-1 Envgp140 trimers elicit neutralizing antibodies without efficient induction of conformational antibodies.

Currently, no vaccine for human immunodeficiency virus (HIV-1) provides protection from virus infection. One reason for these disappointing results has been the difficulty of current vaccine candidates to elicit high-titer, broadly reactive immunity to a large number of viral proteins. Recently, our laboratory demonstrated that the coupling of C3d to a soluble trimerized HIV-1 envelope (Env(gp140(FT))) elicited higher titers of neutralizing antibodies than monomers of Env(gp120) coupled to C3d [Bower JF, Yang X, Sodroski J, Ross TM. Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d. J Virol 2004;78(9):4710-9]. To determine if the induction of conformational antibodies correlated with neutralization, mice (BALB/c) were primed (2x) with DNA plasmids expressing monomeric Env(gp120) or trimeric Env(gp140) alone or fused to mC3d(3) at one of two doses (2.0microg or 0.2microg), followed by a boost of recombinant uncleaved, trimeric Env(gp140). Regardless of the priming dose of DNA, all mice had high-titer anti-Env IgG antibodies. Interestingly, Env(gp140) trimers did not elicit higher titers of antibodies that recognized conformational Env epitopes compared to monomers of Env(gp120). Therefore, additional parameters were examined for correlation with neutralization. For neutralization-resistant HIV-1 isolates, ADA and YU-2, neutralization correlated with high-titer, high avidity antibodies, with Env(gp140) eliciting slightly higher neutralization titers than Env(gp120). In contrast, none of the measured parameters correlated with neutralization for the more neutralization-sensitive isolates, MN or 89.6. Therefore, even though soluble, uncleaved Env(gp140) trimers may be marginally more effective at eliciting neutralizing antibodies than Env(gp120), neutralization does not appear to correlate with the elicitation of conformationally dependent antibodies.

Differences in transport of fatty acids and expression of fatty acid transporting proteins in adipose tissue of obese black and white women.

We have reported that the rate of de novo triglyceride (TG) synthesis by omental, but not subcutaneous, adipose tissue was higher in African-American women (AAW) than in Caucasian women (CAW). The purpose of this study was to explore the potential mechanisms underlying this increase. Toward that end, we determined the activities of key enzymes in the pathway of TG synthesis, the rates of uptake of fatty acids by adipocytes, mRNA and protein levels of the fatty acid-transporting proteins FAT/CD36 and FATP, and mRNA and protein levels of PPARgamma in omental fat of AAW and CAW. The results showed 1) no difference in the activity of phosphofructokinase, glycerol-3-phosphate dehydrogenase, or diacylglycerol acyltransferase; 2) a higher rate of fatty acid uptake by adipocytes of the AAW; 3) an increase in the mRNA and protein levels of CD36 and FATP4 in the fat of the AAW; and 4) an increase in the mRNA and protein levels of PPARgamma, which can stimulate the expression of CD36 and FATP. These results suggest that the increase in the transport of fatty acid, which is mediated by the overexpression of the transport proteins in the omental adipose tissue of the AAW, might contribute to the higher prevalence of obesity in AAW.

C3d enhances immune responses using low doses of DNA expressing the HIV-1 envelope from codon-optimized gene sequences.

DNA vaccinations effectively induce both humoral and cellular immune responses to immunogens from diverse infectious agents. However, DNA vaccines expressing the HIV-1 envelope glycoprotein (Env) are poorly immunogenic when expressed from wild-type (wt) DNA sequences. Two recent approaches used to enhance the immunogenicity of Env expressed from a DNA vaccine are the fusion of the molecular adjuvant, C3d, to a soluble form of Env and the use of codon-optimized (co) env gene inserts. Independently, each approach enhances antibody titer and cellular responses against Env expressed from gene inserts. The goal of this study was to examine if both codon-optimization of env gene inserts and C3d conjugation to Env could function in a synergistic manner to enhance immunogenicity. Mice (BALB/c) were inoculated with decreasing doses (2.0 microg, 0.2 microg or 0.02 microg) of co DNA expressing Env alone or fused to three copies of murine C3d (mC3d3) gene. Mice vaccinated with the highest dose (2.0 microg) of DNA had high anti-Env specific antibody titers regardless of the addition of mC3d3. At lower doses (0.2 microg and 0.02 microg) of DNA, mice vaccinated with Env-mC3d3 had enhanced immune responses compared to mice vaccinated with DNA expressing Env only. In addition, mice vaccinated with Env-mC3d3 at the highest doses of DNA had enhanced interleukin-4 secreting cells, while mice vaccinated with the lowest dose of DNA had enhanced interferon-gamma secreting cells. Therefore, both codon-optimization of env sequences and C3d conjugation to Env appear to enhance anti-Env antibodies in an independent and additive manner.

Unique V3 loop sequence derived from the R2 strain of HIV-type 1 elicits broad neutralizing antibodies.

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies. In this study, DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1(R2) using both wild-type and codon-optimized gene sequences. Three copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein. Mice (BALB/c) vaccinated with DNA plasmid expressing the gp120(R2) using codon-optimized Env sequences elicited high-titer anti-Env antibodies regardless of conjugation to C3d. In contrast, only mice vaccinated with DNA using wild-type gp120(R2) sequences fused to mC3d(3), had detectable anti-Env antibodies. Interestingly, mice vaccinated with DNA expressing gp120(R2) from codon-optimized sequences elicited antibodies that neutralized both homologous and heterologous HIV-1 isolates. To determine if the unique sequence found in the crown of the V3 loop of the Env(R2) was responsible for the elicitation of the cross-clade neutralizing antibodies, the codons encoding for the Pro-Met (amino acids 313-314) were introduced into the sequences encoding the gp120(ADA) (R5) or gp120(89.6) (R5X4). Mice vaccinated with gp120(ADA)-mC3d(3)-DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120(89.6)-mC3d(3)-DNA. Therefore, the use of the unique sequences in the Env(R2) introduced into an R5 tropic envelope, in conjunction with C3d fusion, was effective at broadening the number of viruses that could be neutralized. However, the introduction of this same sequence into an R5X4-tropic envelope was ineffective in eliciting improved cross-clade neutralizing antibodies.

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1.

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

Cutting edge: C3d functions as a molecular adjuvant in the absence of CD21/35 expression.

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35(-/-)) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35(-/-) mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35(-/-) mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d.

DNA vaccines expressing the envelope (Env) of human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting immune responses. Oligomeric or trimeric (gp140) forms of Env that more closely mimic the native proteins on the virion are often more effective immunogens than monomeric (gp120) envelopes. In this study, several forms of Env constructed from the HIV-1 isolate YU-2 (HIV-1(YU-2)) were tested for their immunogenic potential: a trimeric form of uncleaved (-) Env stabilized with a synthetic trimer motif isolated from the fibritin (FT) protein of the T4 bacteriophage, sgp140(YU-2)(-/FT), was compared to sgp140(YU-2)(-) without a synthetic trimerization domain, as well as to monomeric gp120(YU-2). DNA plasmids were constructed to express Env alone or fused to various copies of murine C3d (mC3d). BALB/c mice were vaccinated (day 1 and week 4) with DNA expressing a codon-optimized envelope gene insert, alone or fused to mC3d. Mice were subsequently boosted (week 8) with the DNA or recombinant Env protein. All mice had high anti-Env antibody titers regardless of the use of mC3d. Sera from mice vaccinated with DNA expressing non-C3d-fused trimers elicited neutralizing antibodies against homologous HIV-1(YU-2) virus infection in vitro. In contrast, sera from mice inoculated with DNA expressing Env-C3d protein trimers elicited antibody that neutralized both homologous HIV-1(YU-2) and heterologous HIV-1(ADA), albeit at low titers. Therefore, DNA vaccines expressing trimeric envelopes coupled to mC3d, expressed in vivo from codon-optimized sequences, elicit low titers of neutralizing antibodies against primary isolates of HIV-1.

Cholesteryl ester transfer protein expression prevents diet-induced atherosclerotic lesions in male db/db mice.

OBJECTIVE: Accompanying more atherogenic lipoprotein profiles and an increased incidence of atherosclerosis, plasma cholesteryl ester transfer protein (CETP) is depressed in diabetic obese patients compared with nondiabetic obese counterparts. The depressed levels of CETP in the plasma of diabetic obese individuals may contribute to the development of an atherogenic lipoprotein profile and atherogenesis. We have examined the effect of CETP expression on vascular health in the db/db model of diabetic obesity. METHODS AND RESULTS: Transgenic mice expressing the human CETP minigene were crossed with db/db strain, and 3 groups of offspring (CETP, db, and db/CETP) were placed on an atherogenic diet for 16 weeks. The proximal aorta was then excised and examined for the presence of atherosclerotic plaques. In db mice, 9 of 11 had intimal lesions with a mean area of 26 098+/-7486 microm2. No lesions greater than 1000 microm2 were observed in db/CETP or CETP mice. CETP-expressing mice had lower circulating cholesterol concentrations than db mice. Fractionating plasma lipids by FPLC indicated that the difference in total cholesterol was primarily attributable to differences in VLDL and LDL. CONCLUSIONS: The expression of human CETP in db/db mice prevented the formation of diet-induced lesions, suggesting an antiatherogenic effect of CETP in the context of diabetic obesity.

Ethnic differences in postprandial triglyceride response to a fatty meal and lipoprotein lipase in lean and obese African American and Caucasian women.

The purpose of this study was to determine if there were differences in the expression of lipoprotein lipase (LPL) in African American (AA) and Caucasian (CA) women. LPL mRNA and protein levels were determined in subcutaneous and omental fat of lean and obese subject from the 2 races (4 groups; 12 to 15 subjects/group). LPL mRNA levels of lean AA were not different from the lean CA women in either fat depot. LPL mRNA levels in the subcutaneous fat of the obese AA were higher than those of CA women (1.3 +/- 0.1 v 0.86 +/- 0.06, P.05), but not different in omental fat. LPL mass in subcutaneous fat of lean AA was higher (0.95 +/- 0.09 v 0.64 +/- 0.06, P.05), but not different in omental fat from the CA women. LPL mass in subcutaneous and omental fat was not different in the 2 obese groups. Differences in the activity of LPL were evaluated by (1) measuring the increments of triglycerides (TG) at 2, 4, 6, and 8 hours after a fat-rich meal and (2) by measuring postheparin plasma lipolytic activity. Plasma TG levels in the lean AA were lower than those of the lean CA women at basal and at 2, 4, 6, and 8 hours postprandially. The increase in TG levels at 2 hours tended to be lower in the AA than the CA women, was significantly lower at 4 hours (24 +/- 5 v 45 +/- 7, P.05), and was not different 8 hours postprandially. No differences were observed in either the absolute or the incremental concentrations of TG in the obese groups. Postheparin plasma LPL activity was higher in the lean AA than the lean CA women (4.8 +/- 0.4 v 3.4 +/- 0.4, P.05), but not different in the obese groups. These results indicate that the lower TG concentrations in the lean AA women may be partly due to enhanced expression, activity, and intravascular availability of LPL. Furthermore, it appears that the racial differences in expression and function of LPL are attenuated with obesity.