Surface display of major capsid protein on Bacillus subtilis spores against largemouth bass virus (LMBV) for oral administration.

Largemouth bass virus (LMBV) infections resulting in enormous loss are becoming an increasing problem in the largemouth bass industry. Oral vaccination is considered to be an effective and economical measure because of the advantages of non-invasion, no size limitation, lower cost and easily-operated. Based on Bacillus subtilis (B. subtilis) spores, this study successfully constructed the CotC-LMBV recombinant B. subtilis spores and its protective efficacy and immune responses were evaluated. After challenged, the survival rate of largemouth bass orally vaccinated with CotC-LMBV spores was 53.3% and the relative percent survival (RPS) was 45.0% compared to the PBS group. In addition, the specific IgM level in serum in the CotC-LMBV group was significantly higher than in the control groups. In the spleen, the immune-related genes expression detected by quantitative real-time PCR (qRT-PCR) exhibited an increasing trend in different degrees in the CotC-LMBV group, suggesting that innate and adaptive immune responses were activated. This study indicated that oral administration of CotC-LMBV recombinant spores could stimulate an effective immune response and enhance fish immunity against LMBV infection. Therefore, oral vaccination could be an effective approach for the prevention of largemouth bass virus disease.

STAT3-induced NCK1 elevation promotes migration of triple-negative breast cancer cells via regulating ERK1/2 signaling.

BACKGROUND: Noncatalytic region of tyrosine kinase 1 (NCK1) plays a key role in extracellular matrix degradation, which is required for the metastasis of triple-negative breast cancer (TNBC). However, the role NCK1 plays in the metastatic progression of TNBC is unknown. METHODS AND RESULTS: Based on online databases, NCK1 was found to be highly expressed in TNBC as compared to normal breast-like subjects, which was also confirmed using TNBC cells and a tissue microarray. NCK1 expression gradually decreased with increased tumor stage. High NCK1 expression displayed a poor prognosis in lymph node-positive metastatic TNBC patients, but not in lymph node-negative patients. Using transwell assays and immunoblotting, we confirmed that NCK1 overexpression promoted, while NCK1 downregulation inhibited migration capabilities, as well as the expression of matrix metalloproteinases (MMP2/9), uridylyl phosphate adenosine, and plasminogen activator inhibitor-1 in TNBC cells. Mechanistically, NCK1 upregulation mediated the activation of MMP2/9 through ERK1/2 activity. Signal transducer and activator of transcription 3 (STAT3) was positively correlated with NCK1. STAT3 could directly bind to the promoter region of NCK1 to promote its expression and was accompanied by the elevation of MMP2/9 and ERK1/2 signaling, which were partially abolished by the knockdown of NCK1 in TNBC cells. CONCLUSIONS: NCK1 may serve as a diagnostic and prognostic marker of metastatic TNBC. STAT3 upregulation promoted the expression of NCK1, which subsequently induced the migration and activity of MMPs in a ERK1/2 signaling-dependent manner in TNBC cells. NCK1 is a promising target for improving TNBC migration.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

A rate-limiting role for Dickkopf-1 in bone formation and the remediation of bone loss in mouse and primate models of postmenopausal osteoporosis by an experimental therapeutic antibody.

Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies.

Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC), but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

Safety and immunogenicity of the Merck adenovirus serotype 5 (MRKAd5) and MRKAd6 human immunodeficiency virus type 1 trigene vaccines alone and in combination in healthy adults.

Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of < or = 18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (< or = 200 or >200) and Ad6 titers (< or = 18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to > or = 2 vaccine antigens were 14%, 63%, and 71% at 10(9), 10(10), and 10(11) viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 10(10) vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.

Vaccine-induced cellular responses control simian immunodeficiency virus replication after heterologous challenge.

All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

IgG2m4, an engineered antibody isotype with reduced Fc function.

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.

Short communication: In vitro synergy between peptides or neutralizing antibodies targeting the N- and C-terminal heptad repeats of HIV Type 1 gp41.

Class 1 and class 2 fusion peptides bind to the trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of HIV-1 envelope glycoprotein gp41, respectively, and block its intramolecular folding required for Env-mediated viral and host cell membrane fusion and subsequent viral entry. Using a combination of T-20 (class 1) and (CCIZN17)(3) (class 2), we provide evidence that these classes of fusion peptides work synergistically in an in vitro infectivity assay in inhibiting the entry of primary HIV-1 isolate 89.6 with combination indexes reaching 0.37 and 0.32 at IC(50) and IC(90), respectively. We further demonstrate a similar degree of neutralization synergy between a monoclonal antibody (MAb), D5, targeting the hydrophobic pocket region of the NHR, and 2F5, a well-characterized MAb that targets the C-terminal end of CHR and the membrane-proximal external region (MPER), providing a rational basis for developing combination vaccines targeting these two highly conserved regions of gp41.

The effect of early versus delayed challenge after vaccination in controlling SHIV 89.6P infection.

We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A*01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p<0.0001). After week 12, no further decay was observed. Twenty of 24 vaccinated animals maintained more CD4+ T cells and kept their viral load at least one order of magnitude lower than the control animals throughout the chronic phase of the study. All 24 vaccinated animals survived the duration of the study. The viral and T cell kinetics over the first two weeks differed between the vaccinated groups, with more recent vaccination improving the early control of virus (p-value=0.027). The rates of virus specific CD8+ T cell expansion were greater in animals having higher viral loads at one week (r=0.45, p=0.029), suggesting that the kinetics of early viral load may have a role in virus specific CD8+ T cell generation, although these early differences did not lead to different clinical outcomes within the vaccinated animals.

Quantifying the titer and quality of adenovirus stocks.

The broad application of recombinant adenoviruses to the development of vaccines and gene therapy vectors has encouraged the development of molecular assays for the facile quantitation of adenoviral particles and the assignment of their infectious potency. The Genome Quantitation Assay (GQA) and the QPCR-Based Potency Assay (QPA) developed for adenoviruses offer the attributes of precision, rapidity, and high throughput either performed manually or facilitated by simple automated liquid handling systems. These assay attributes allow for accelerated process development support and product characterization and release. The assays for adenovirus could offer the additional advantage in that their quantitation is based on viral replication independent of cytopathology permitting quantitation of serotypes that cause minimal cytopathic effect (CPE) in 293 cells and specificity that allows the components of multivalent vaccines to be discriminated and quantitated for release.

Application of PCR technology in vaccine product development.

The complex process of vaccine product development needs to be tightly controlled and closely monitored to ensure vaccine quality and consistency. Since its inception, PCR has been widely used in all stages of vaccine product development as a tool to assist in the evaluation of vaccine quality, safety and efficacy. In this review, the general principles of conventional and real-time quantitative PCR (Q-PCR) technology and its application in vaccine product development for quantitation of vaccine dose (genome quantitation assay), infectivity (Q-PCR-based potency assay), process residuals, stability, adventitious agents, safety assessment and clinical studies are described. The future outlook and the advantages and disadvantages of this technology are also discussed.

Vaccine-induced cellular immune responses reduce plasma viral concentrations after repeated low-dose challenge with pathogenic simian immunodeficiency virus SIVmac239.

The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4(+) memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.