2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

Lipoprotein Drug Delivery Vehicles for Cancer: Rationale and Reason.

Lipoproteins are a family of naturally occurring macromolecular complexes consisting amphiphilic apoproteins, phospholipids, and neutral lipids. The physiological role of mammalian plasma lipoproteins is to transport their apolar cargo (primarily cholesterol and triglyceride) to their respective destinations through a highly organized ligand-receptor recognition system. Current day synthetic nanoparticle delivery systems attempt to accomplish this task; however, many only manage to achieve limited results. In recent years, many research labs have employed the use of lipoprotein or lipoprotein-like carriers to transport imaging agents or drugs to tumors. The purpose of this review is to highlight the pharmacologic, clinical, and molecular evidence for utilizing lipoprotein-based formulations and discuss their scientific rationale. To accomplish this task, evidence of dynamic drug interactions with circulating plasma lipoproteins are presented. This is followed by epidemiologic and molecular data describing the association between cholesterol and cancer.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

HER2/HER3 regulates lactate secretion and expression of lactate receptor mRNA through the MAP3K4 associated protein GIT1.

One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Downregulation of GLUT4 contributes to effective intervention of estrogen receptor-negative/HER2-overexpressing early stage breast disease progression by lapatinib.

Tamoxifen and aromatase inhibitors (AIs) have shown efficacy in prevention of estrogen receptor-positive (ER+) breast cancer; however, there exists no proven prevention strategy for estrogen receptor-negative (ER-) breast cancer. Up to 40% of ER- breast cancers have human epidermal growth factor receptor 2 overexpression (HER2+), suggesting HER2 signaling might be a good target for chemoprevention for certain ER- breast cancers. Here, we tested the feasibility of the HER2-targeting agent lapatinib in prevention and/or early intervention of an ER-/HER2+ early-stage breast disease model. We found that lapatinib treatment forestalled the progression of atypical ductal hyperplasia (ADH)-like acini to ductal carcinoma in situ (DCIS)-like acini in ER-/HER2+ human mammary epithelial cells (HMECs) in 3D culture. Mechanistically, we found that inhibition of HER2/Akt signaling by lapatinib led to downregulation of GLUT4 and a reduced glucose uptake in HER2-overexpressing cells, resulting in decreased proliferation and increased apoptosis of these cells in 3D culture. Additionally, our data suggest that HER2-driven glycolytic metabolic dysregulation in ER-/HER2+ HMECs might promote early-stage breast disease progression, which can be reversed by lapatinib treatment. Furthermore, low-dose lapatinib treatment, starting at the early stages of mammary grand transformation in the MMTV-neu* mouse model, significantly delayed mammary tumor initiation and progression, extended tumor-free survival, which corresponded to effective inhibition of HER2/Akt signaling and downregulation of GLUT4 in vivo. Taken together, our results indicate that lapatinib, through its inhibition of key signaling pathways and tumor-promoting metabolic events, is a promising agent for the prevention/early intervention of ER-/HER2+ breast cancer progression.

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.

Recommendations on incurred sample stability (ISS) by GCC.

The topic of incurred sample stability (ISS) has generated considerable discussion within the bioanalytical community in recent years. The subject was an integral part of the seventh annual Workshop on Recent Issues in Bioanalysis (WRIB) held in Long Beach, CA, USA, in April 2013, and at the Global CRO Council for Bioanalysis (GCC) meeting preceding it. Discussion at both events focused on the use of incurred samples for ISS purposes in light of results from a recent GCC survey completed by member companies. This paper reports the consensus resulting from these discussions and serves as a useful reference for depicting ISS issues and concerns, summarizing the GCC survey results and providing helpful recommendations on ISS in the context of bioanalytical method development and application.

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.

Racial difference in Acylation Stimulating Protein (ASP) correlates to triglyceride in non-obese and obese African American and Caucasian women.

BACKGROUND: Acylation Stimulating Protein (ASP) has been shown to influence adipose tissue triglyceride (TG) storage. The aim was to examine ethnic differences in ASP and leptin levels in relation to lipid profiles and postprandial changes amongst African American (AA) and Caucasian American (CA) women matched for BMI. METHODS: 129 women were recruited in total (age 21 - 73 y): 24 non-obese (BMI < 30 kg/m²) CA, 27 obese (BMI ≥ 30 kg/m²) CA, 13 obese diabetic CA, 25 non-obese AA, 25 obese AA, and 15 obese diabetic AA. Cholesterol, HDL-C, LDL-C, apoB, glucose and insulin were measured at baseline. TG, non-esterified fatty acids, leptin, and ASP were measured at baseline and postprandially following a fat meal. RESULTS: ASP, leptin, insulin and TG were significantly increased in obese subjects within each race. However, AA women had significantly lower ASP and TG than CA women at all BMI. Obese and diabetic AA women had significantly lower apoB levels than CA women when compared to their respective counterparts. For AA women, fasting ASP was positively correlated with BMI, cholesterol, apoB, LDL-C and glucose. For CA women, fasting ASP was positively correlated with BMI, leptin, glucose and insulin. However, for any given BMI, ASP was significantly reduced in AA vs CA (p = 0.0004). Similarly, for any given leptin level or TG levels, ASP was significantly lower in AA women (p = 0.041 and p = 0.003, respectively). CONCLUSION: CA women have higher baseline TG levels and an earlier TG peak that is accompanied with higher ASP levels suggesting increased ASP resistance, while AA women have lower baseline TG levels and a later TG peak at lower ASP levels suggesting increased ASP sensitivity. This may explain why AA women may have fewer metabolic complications, such as diabetes and CVD, when compared to their Caucasian counterparts at the same level of obesity.

Solve the succession crisis by growing inside-outside leaders.

In his analysis of 1800 successions, Harvard Business School professor Bower found that companies performed significantly better when they appointed insiders to the job of CEO. Other researchers, including Jim Collins in Good to Great, have come to similar conclusions working from different data sets. Yet Bower finds far too many companies have no succession plans; as a result, when the time comes to name a new chief executive, more firms turn to outsiders. Both insider and outsider CEOs have strengths and weaknesses at the start. Insiders know the company and its people but are often blind to the need for radical change. Outsiders see the need for a new approach but can't make the necessary changes because they don't know the organization or industry sector well enough. What companies must do, then, is find a way to nurture what Bower calls inside-outsiders--internal candidates who have outside perspective. Often such executives have spent much of their time away from the mainstream of the organization, and away from headquarters, living with new opportunities and threats. Before becoming CEO, Procter & Gamble's A.G. Lafley, for instance, worked for years building P&G's Chinese cosmetics operation rather than the core detergent business. IBM's Sam Palmisano was a champion of software and open systems at a time when Big Blue was essentially a closed-system, hardware-oriented company. Nascent inside-outsiders should enter the CEO-training process by the time they are 30 and be given the opportunity to manage a whole business, so that they become good insiders. But they also need to be mentored with an eye toward preserving their outsider perspective, so they learn how to turn their new ideas into great businesses and are protected from old-timers who might be inclined to teach them a lesson.

How Managers' everyday decisions create or destroy your company's strategy.

Senior executives have long been frustrated by the disconnection between the plans and strategies they devise and the actual behavior of the managers throughout the company. This article approaches the problem from the ground up, recognizing that every time a manager allocates resources, that decision moves the company either into or out of alignment with its announced strategy. A well-known story--Intel's exit from the memory business--illustrates this point. When discussing what businesses Intel should be in, Andy Grove asked Gordon Moore what they would do if Intel were a company that they had just acquired. When Moore answered, "Get out of memory," they decided to do just that. It turned out, though, that Intel's revenues from memory were by this time only 4% of total sales. Intel's lower-level managers had already exited the business. What Intel hadn't done was to shut down the flow of research funding into memory (which was still eating up one-third of all research expenditures); nor had the company announced its exit to the outside world. Because divisional and operating managers-as well as customers and capital markets-have such a powerful impact on the realized strategy of the firm, senior management might consider focusing less on the company's formal strategy and more on the processes by which the company allocates resources. Top managers must know the track record of the people who are making resource allocation proposals; recognize the strategic issues at stake; reach down to operational managers to work across division lines; frame resource questions to reflect the corporate perspective, especially when large sums of money are involved and conditions are highly uncertain; and create a new context that allows top executives to circumvent the regular resource allocation process when necessary.

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.