Standard Clinical Pathology Research Articles

257 (PB-081) Poster - Development and Validation of a Digital-Artificial Intelligent (AI) enabled Assay to predict early-stage breast cancer recurrence

Background: Breast cancer (BC) is the most frequently diagnosed cancer worldwide and the second leading cause of cancer-related deaths. To advance predictive recurrence models we developed an AI-image analysis platform that utilizes whole slide images (WSI) to phenotype invasive BC (IBC) at the tissue-cell architectural level. The objective was to produce an AI-IBC phenotype that includes a novel methodology to grade BC along with additional features that reflect biological pathways. We sought to combine these extracted and complex image feature sets with standard clinical pathology data to develop readily accessible models that predict early-stage BC recurrence.

No Correlation Between Presenting Symptoms Prior to Suburethral Sling Removal and Explanted Pathology Findings Suggests No Need for Routine Microscopic Pathology Evaluation.

We compared explanted midurethral sling (MUS) standard clinical pathology report findings with presenting symptoms before synthetic sling removal (SSR). An institutional review board-approved, prospectively maintained database of women who underwent a single SSR for MUS-related complication(s) was retrospectively reviewed for demographics, time interval between MUS placement and SSR, MUS type, and presenting symptoms before SSR. We compared 2 groups, one with MUS-related symptoms, including incontinence, voiding dysfunction, dyspareunia/pain, erosion, exposure, and/or urinary tract infections, and the second group characterized by self-reported symptoms attributed to the mesh material (systemic, autoimmune, or inflammatory disorder-mesh reaction group). From 2005 to 2019, 332 of 473 women met study criteria, with 24 reporting mesh reaction-related symptoms. There was no difference in microscopic pathology findings between the 2 groups. In the mesh-related group, women were younger (P = 0.041) and had more pain/dyspareunia as their primary SSR indication (P = 0.002). That group was also significantly more likely to have a preexisting diagnosis of fibromyalgia (P < 0.001) and established autoimmune disorder (P = 0.033). No significant correlation between presenting findings and sling pathology was observed. No correlation was observed when comparing pathological findings of explanted MUS with presenting symptoms of MUS-related complications, including in women with mesh reaction-related concerns.

A Novel Next-Generation FVIII Mimetic, Mim8, Has a Favourable Safety Profile. Results from a 26-Week Safety Study in Cynomolgus Monkeys

Introduction Mim8 is in clinical development as a novel, next generation FVIII mimetic for subcutaneous prophylactic treatment for people with Hemophilia A (HA) with and without inhibitors. Mim8 is a fully human bispecific antibody bridging FIXa and FX. Mim8 showed approximately 15-fold higher potency compared to a sequence identical analogue (SIA) of the FVIII mimetic emicizumab (Hemlibra®) in thrombin generation assays (TGAs) and in vivo in mouse bleeding models (Kjellev et al., 2019; Ley et al. 2020). Mim8 binds to cynomolgus monkey and human FIX and FX with comparable affinities, and the pharmacological activity of Mim8 in cynomolgus monkeys was demonstrated both in vitro in TGA and in vivo in a cynomolgus bleeding model (Ley et al., 2020). The present study examined the safety and ex vivo pharmacodynamic effects of Mim8 in cynomolgus monkeys after weekly dosing for 26 weeks. Material and Methods Sexually mature cynomolgus monkeys were dosed subcutaneously (SC) with Mim8 at dose-levels of 0, 0.3, 1 or 3 mg/kg/week (4 males and 4 females at each dose-level) for 26 weeks. These dose-levels were chosen based on a previous 13-week safety study showing histopathological changes consistent with exaggerated pharmacology in a few animals dosed ≥6 mg/kg/week. During the study, the animals were monitored clinically and blood was sampled for measurement of standard clinical pathology, Mim8 exposure, anti-drug antibodies (ADA). The pharmacodynamic activity was assessed by ex vivo TGA in plasma under antibody-induced HA-like conditions and measurement of activated partial thromboplastin time (aPTT). After 26 weeks of exposure, the animals were euthanised, followed by macroscopic and microscopic pathological examination on all major organ systems. Results Weekly dosing with Mim8 at dose-levels up to 3 mg/kg/week for 26 weeks was well tolerated and the macroscopic and microscopic pathological examinations revealed no thrombi or other signs of excessive coagulation activation. In accordance, no significant changes in platelet counts, fibrinogen concentration or prothrombin time were observed. A pharmacodynamic effect was observed in dosed animals as a dose-dependent increase in ex vivo thrombin generation (TG; Figure 1) and shortening of aPTT. The steady state exposure increased with increasing dose level in a dose-proportional manner, reaching concentrations at least 10-fold above the expected clinical concentrations. However, as expected when dosing a human antibody to monkeys, ADAs developed in approximately half of the animals at all dose levels, leading to lack of exposure and reversal of the aPTT and TG towards the predose levels. Conclusion In conclusion, administration of up to 3 mg/kg/week Mim8 to cynomolgus monkeys for 26 weeks showed relevant pharmacodynamic effect and was well tolerated with no observation of thrombi or other signs of excessive coagulation activation, providing a favourable safety ratio to expected clinical exposure levels. Mim8 is currently being evaluated in a clinical Phase 1/2 trial. References Kjellev SL et al. (2019) Mim8 - a next-generation FVIII mimetic bi-Specific antibody - potently restores the hemostatic capacity in hemophilia a settings in vitro and in vivo. Blood, 134 (Supplement 1): 96. Ley CD et al. (2020) Improved effect of Mim8, a next-generation FVIII mimetic, translates from human in vitro to humanized mouse and cynomolgus models. European Association for Haemophilia and Allied Disorders (EAHAD 2020). Haemophilia, 26 (S2), P028. Disclosures Kjalke: Novo Nordisk A/S: Current Employment, Current equity holder in publicly-traded company.

Novel methods for the assessment of safety pharmacology and toxicology parameters in anesthetized and ventilated dogs receiving inhaled drugs

IntroductionFor nonclinical drug development, it is optimal if safety pharmacology and toxicology studies are performed in a model that reasonably represents the patient the drug is intended to treat. To simulate prolonged inhalation therapy in ventilated patients, GLP inhalation toxicology methods, including safety pharmacology endpoints, in anesthetized, intubated and mechanically ventilated dogs were developed. This model required establishment of a canine intensive care unit (ICU) capable of providing prolonged anesthesia (propofol infusion and morphine titration) and partial parenteral nutrition (dextrose, amino acids and lipids) while safety parameters were monitored. MethodTelemetry was used to continuously monitor heart rate, ECG and blood pressure. Blood gas parameters were periodically measured while oxygen saturation and core temperature were reported continuously. Glucose was measured hourly while other standard clinical pathology (hematology, coagulation, clinical chemistry) samples were evaluated approximately every 12 h. Aerosols were administered continuously over 48 h by inhalation using a mesh nebulizer (Aerogen Solo) fed by a syringe pump into a humidified circuit of a critical care ventilator (LTV® 1000) ending in an endotracheal tube placed in the trachea. Animals were ventilated with pressure control ventilation targeting a respiratory minute volume of 2.0–3.5 l per minute (LPM). Peak inspiratory pressure (PIP) was maintained between 10 and 17 cm H2O and inspiratory time was set to 1 s with an inspiratory:expiratory (I:E) ratio of 1:2. Ventilator parameters and anesthesia were adjusted to maintain normal PaCO2 levels and adequate sedation, respectively. Novel methods were developed to determine dose and particle size in vitro as on-line measurements were not feasible during in vivo aerosol delivery. Results and discussionAcceptable baseline measurements were established for all parameters over the 48-h evaluation period, qualifying the method as appropriate for assessment of GLP safety pharmacology and toxicology studies.

Catha edulis-induced skeletal muscle toxicity in experimental rats via regulation of rhabdomyolysis biomarkers

Background: Recently, there are clinical reports on the potential of Catha edulis (khat) to induce muscular toxicity. C. edulis (khat) is an evergreen shrub and a well-known controversial plant due to the content of natural stimulant, cathine and cathinone. Objective: The main objective of the study is to evaluate the possible effect of C. edulis leaves extract to induce rhabdomyolysis in vivo. Materials and Methods: Sprague Dawley rats were grouped and treated with khat extract at two different doses (250 and 500 mg/kg), while atorvastatin was used as positive control for 28 days. Body weight was measured throughout the study period. Overnight urine was collected from each rat at the 28th day for myoglobin (Myo) analysis. Terminal blood samples were collected from sacrificed animals for the measurement of serum biomarkers and clinical chemistry. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum creatinine (CR) were monitored. Skeletal muscle, cardiac muscle, and kidney were collected for histopathological examination. Results: Animals received 250 mg/kg khat extract had shown mild-to-no gait disorders, while at higher dose extract (500 mg/kg) had reduced the body weight of rats with marked increase of gait disorders compared to control. CR, AST, and ALT were elevated in high-dose administration and in rats received ethanol. The tested biomarkers such as heart-type fatty acid-binding protein (HFABP) 3, Troponin I Type 1 slow skeletal (TNNI1), and Myo were significantly increased in khat high dose and statin treatment, but not in low-dose extract and alcohol. The increase in HFABP and TNN1 results were well reflected in histopathological findings of skeletal myofiber degeneration and in the hemorrhages and pyknosis of nucleus observed in the cardiac muscle. Conclusion: These results provide evidence that khat chewing contributes to the development of muscle toxicity and probable rhabdomyolysis. The current subject thus warrants detailed studies which could emphasize on the cardiac complications and muscular toxicity mechanisms.

OPTIMATION OF PLATELET RICH PLASMA PRODUCTION PROTOCOL USING DOUBLE CENTRIFUGATION METHODS

Currently, therapy with Platelet Rich Plasma (PRP) has been widely used and continues to grow for various clinical applications. Along with its development, there are various options in the method of obtaining PRP, either automatic or manual, while one of the most reliable according to the literature is a double centrifugation method. The purpose of this research is to produce an optimization of the double centrifugation method. This study uses experimental data obtained by researchers at the Clinical Pathology Laboratory of Dr. Soetomo Hospital, Surabaya. Experiments generally were conducted on stored blood obtained from the blood bag from PMI with CPD anticoagulant. Optimization of double centrifugation method to produce PRP with optimum platelet count could be obtained by using an "I"-shaped tube as well as other specialized equipment which are currently not available in standard clinical pathology laboratory, and also fresh blood. The resulting increase amounted to 4.11 times with the platelet count of 1.152 million.

Biomarker evaluation of skeletal muscle toxicity following clofibrate administration in rats

The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.

IMPS-02EXPRESSION OF MULTIPLE ANTIGENS IN INDIVIDUAL GLIOBLASTOMA PATIENT SAMPLES EVALUATED BY IMMUNOCHEMISTRY

Cellular heterogeneity is a hallmark characteristic of ostensibly similar cell populations occurring during neural and glial development, and during tumorigenesis and tumor progression. For the latter case, large collections of formalin-fixed paraffin embedded (FFPE) clinical specimens are a valuable resource for evaluating gene expression across large study cohorts. Many of these patient samples consist of serial tumor sections each stained for a single antigen by chromogenic immunohistochemistry (IHC), as is standard clinical pathology practice. Quantifying multiple tumor related antigens in the context of GBM tissue is an essential step towards understanding the fundamental biology of these tumors as well as developing novel targeted therapies. Towards this end, we have developed methods for combining semi-quantitative analysis of IHC processed tumor sections with regional annotation by a clinical pathologist. This has allowed us to consider patterns of antigen co-expression as well as spatial relationships with tumor structures and physiological status. We will present our observations on expression of tumor-associated antigens IL13Rα2, HER2 and EGFR, and associations with proliferation as indicated by Ki67 expression and the vasculature as marked by the endothelial cell marker CD34.

Assessment Criteria for Accreditation of Government Hospitals' Laboratories in Sudan according to the international standards. Khartoum State – Sudan.

Introduction: The international standard Clinical Pathology Accreditation UK , WHO-AFRO accreditation and ISO 15189 Medical laboratories -Particular requirements for quality and competence are the basis for this accreditation. These requirements not only require a management system and manual in the medical laboratory but also require that the laboratory be found competent to perform specific medical tests or types of tests. Objective: The study aimed to assessment Governmental Hospitals’ Laboratories situation and their performance according to international standards scale. Methods: The present study designed descriptive Series of case studies in thirty eight Government Hospitals’ Laboratories located in Khartoum state, the intervention application of Continuous Quality Improvement techniques to assess laboratories processes; total Quality Systems standards into laboratories were measured by Checklist contain standards elements and score of each element was conducted according to their importance of international standards. The quality control in this study was carried out for 15 tests, used control sera. Results: Results availability of international standards for total quality management implementation in the laboratories is between 36% -86% the mean 65%, the safe laboratory design and organization 77.5%, laboratory organization 48.5%, document and management system 45.5%, quality of Personnel management 55.5%. The observed errors of laboratories in the preanalytical phase of testing were 60% and 37% in analytical phase, only 3% errors was reported in the post-analytical phase, continual improvement for laboratories auditing 26% . The quality control program 67% had internal quality control, 33% had national quality control, and the acceptability of quality control results for all laboratories was 72%, total absolute error 55.56%, inaccuracy (variation) 11.5%, imprecision CV 25.1%. Conclusion: This study showed significant correlation between adoption of international standards and improved of accuracy and reliability on medical laboratories tests.

Essential variability in standard clinical pathology biomarkers in beagle dogs instrumented with chronic indwelling telemetry devices

Essential variability in standard clinical pathology biomarkers in beagle dogs instrumented with chronic indwelling telemetry devices

A Multiplex Two-Color Real-Time PCR Method for Quality-Controlled Molecular Diagnostic Testing of FFPE Samples

BackgroundReverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.MethodsWe developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.ResultsReagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R2>0.99), signal-to-analyte response (slope 1.0±0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.ConclusionsThis quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.

Background variability in standard clinical pathology biomarkers in beagle dogs instrumented with chronic indwelling telemetry devices

IntroductionContemporary best practice recommendations in preclinical cardiovascular safety assessment promote 3Rs principles. This includes the employment of within-subjects experimental designs to evaluate discrete, acute doses of investigational new drugs, as well as the maintenance of stock colonies of appropriate large animal test systems. Such colony species are often tested repeatedly on independent studies with provision of appropriate recovery periods and requisite health status evaluations (e.g., physical examinations, electrocardiographic assessments, clinical pathology evaluations). MethodsTo investigate the utility of the often reiterative process of pre- or inter-study clinical pathology testing to help ascertain health status of non-naïve, telemetered canines (beagle dogs), the present study collated the results of a randomly selected set of animals approximately every three months for a period of three years. ResultsAlthough occasionally a few routine hematology or clinical chemistry endpoints did demonstrate evidence of systematic trending over time, none of the observed fluctuations fell outside the range of expected biological variability, nor would have prevented assignment of any given animal to study. DiscussionThe present findings illustrate a high degree of consistency in routinely assessed clinical pathology parameters during the course of chronic telemetry instrumentation in the canine, including relative to historical control data in healthy, experimentally naïve animals of the same species and source, maintained under analogous laboratory conditions. The data suggest that routine assessment of such parameters for the purposes of facilitating judgments concerning suitability for study may represent a pursuit of little overall value, and which may be reasonably accomplished based on alternative, observation-based screening procedures.

Assessment of renal injury in vivo

The ICH S7A (Safety Pharmacology for Human Pharmaceuticals) guidelines specify that potential adverse pharmacologic effects of a test substance on renal function should be evaluated in supplemental studies when there is a cause for concern (ICH, 2001). For the most part, this can easily be accomplished by examination of the appropriate analytes in urine and blood collected as part of the routine preclinical safety studies. This review will serve as an overview of the selection, interpretation and limitations of standard clinical pathology methods (serum chemistry and urinalysis) for assessment of renal function in such studies, as well as provide some information on emerging biomarkers of renal function.

A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia.

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10(-1), and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

Two-Generation Reproduction Study and Immunotoxicity Screen in Rats Dosed with Phenol via the Drinking Water

This study evaluated the potential reproductive toxicity of phenol in a rat two-generation reproduction study, which included additional study endpoints, such as sperm count and motility, developmental landmarks, histological evaluation of suspect target organs (liver, kidneys, spleen, and thymus), weanling reproductive organ weights, and an immunotoxicity screening plaque assay. Phenol was administered to 30 Sprague-Dawley rats/sex/group in the drinking water at concentrations of 0, 200, 1000, or 5000 ppm. Parental (P1) animals were treated for 10 weeks prior to mating, during mating, gestation, lactation, and until sacrifice. The F1 generation (P1 offspring) was treated using a similar regimen, while the F2 generation was not treated. After mating, 10 P1 males/group were evaluated using standard clinical pathology parameters and an immunotoxicity screening plaque assay. Significant reductions in water and food consumption were observed in the 5000-ppm group in both generations; corollary reductions in body weight/body weight gain were also observed. Mating performance and fertility in both generations were similar to controls, and no adverse effects on vaginal cytology or male reproductive function were observed. Vaginal opening and preputial separation were delayed in the 5000-ppm group, and were considered to be secondary to the reduction in F1 body weight. Litter survival of both generations was reduced in the 5000-ppm group. Absolute uterus and prostate weights were decreased in the F1 generation at all dose levels; however, no underlying pathology was observed and there was no functional deficit in reproductive performance. Therefore, these findings were not considered to be adverse. No evidence of immunotoxicity was noted in the 5000-ppm group. The effects noted at the high concentration were presumed to be associated with flavor aversion to phenol in the drinking water. Based on a comprehensive examination of all parameters, the no-observable-adverse-effect level (NOAEL) for reproductive toxicity of phenol administered in drinking water to rats is 1000 ppm. The corresponding daily intake of phenol for an adult rat at the NOAEL of 1000 ppm is equivalent to about 70 mg/kg/day for males and 93 mg/kg/day for females.

Bethesda conference: conference for the design of clinical trials to study circulatory support devices for chronic heart failure

James M. Anderson, MD Eugene H. Blackstone, MD Harvey S. Boroevetz, PhD Arthur Ciarkowski, MD Jack G. Copeland III, MD Maria Rosa Costanzo-Nordin, MD Kurt Daase, MD Mary Amanda Dew, PhD Michael J. Domanski, MD Benjamin H. Eidleman, MD Roger W. Evans, PhD David J. Farrar, PhD O. H. Frazier, MD Bartley P. Griffith, MD Charles-Julien Hahn, MD J. Donald Hill, MD Sharon Hunt, MD Kay Kendell, RN Robert L. Kormos, MD Howard R. Levin, MD Donna M. Mancini, MD Patrick M. McCarthy, MD Dennis McNamara, MD Fr Kevin O’Rourke, PhD Lisa Parker, PhD D. Glenn Pennington, MD Victor L. Poirier, PhD Peer M. Portner, PhD Eric A. Rose, MD Wolf Sapirstein, MD Eleanor Schron, MD Rhona Shanker, MD Ramiah Subramanian, MD John Wallwork, MD Lynne Warner Stevenson, MD

Avaliação da função renal através da densidade urinaria e dosagem sérica de uréia e creatinina na aflatoxicose experimental em cães

O presente trabalho refere-se a determinação da densidade urinária e dosagem sérica de uréia e creatinina em cães submetidos a aflatoxicose experimental. Foram utilizados 14 cães (7 machos e 7 fêmeas), sem raça definida, adultos e peso médio de 6,7 ± 1,96kg. Em um período pré-experimento foram realizadas 3 colheitas de urina e sangue de cada animal para avaliação da função renal e as médias das determinações foram utilizadas como controle. Os cães receberam 6 7µg/kg/dia de aflatoxina, via oral, durante 9 dias consecutivos. A partir do quinto dia foram realizadas 4 colheitas com intervalos de 5 dias. Os resultados obtidos demonstraram, através dos sinais clínicos e das lesões macro e microscópicas, o desenvolvimento de aflatoxicose, sendo que não houve alteração na função renal detectável através dos parâmetros estudados.

Immunogenicity and toxicity testing of an experimental HIV-1 vaccine in nonhuman primates.

A highly purified saponin from Q. saponaria (QS-21) was tested in juvenile rhesus macaques for adjuvant activity and toxicity. The QS-21 was tested alone or as part of an experimental subunit HIV-1 vaccine containing a truncated recombinant HIV-1 envelope protein (gp160D) adsorbed to alum. Antibody responses were measured using ELISA and cell-mediated immunity was measured using cellular proliferation assays. Potential toxicity was monitored by standard clinical pathology testing using peripheral blood and urine samples. No toxic effects were observed, even after the administration of the experimental vaccines three times at monthly intervals. The QS-21 saponin adjuvant enhanced total antibody production levels by greater than 100-fold and broadened the specificity of the response so that additional epitopes were recognized, when compared with alum-adsorbed HIV-1 gp160D formulation. Low-level, antigen-specific proliferative responses to HIV-1 recombinant gp160 were induced by either vaccine formulation. Proliferative responses were induced by a sham challenge with soluble recombinant HIV-1 gp160 for all of the animals that had been vaccinated. However, those that received the HIV-complete vaccine formulation containing QS-21 responded significantly better. These data demonstrated that the QS-21 adjuvant augmented both antibody responses and cell-mediated immunity and established immunological memory. The potent adjuvant activity and lack of toxicity suggest that this adjuvant should be safe and effective for use in HIV-1 vaccines.