Polyvalent Conjugate Research Articles

The Safety of 4-valent Meningococcal Conjugate Vaccine in Two Years Old Children: Prospective Cohort Study in Sakhalin Region

Background. Acceptability of routine immunization with vaccine to meningococcus serogroups A, C, W, Y in Russian children have not been studied earlier. Objective. The aim of the study was to understand the safety of polyvalent meningococcal conjugate vaccine administration in two years old children in actual clinical practice. Methods. The vaccination of two years old children against meningococcal disease has been performing in Sakhalin region since April 2018. All children who received single 4-valent meningococcal conjugate vaccine to A, C, W, Y serogroups in the period from April to November 2018 were included into the research. The vaccine safety in general group was estimated by local pediatricians visiting due to general and local adverse effects on vaccine injection in one month period. Safety study for children vaccinated in the period from September to October 2018 was carried out by questioning parents. Results. 1250 children were vaccinated during this research. Local paediatricians has recorded 11 (0.9%) appointments due to adverse effects within 1 month after vaccination. All of them were mild: infiltrates in the vaccine injection site (4 cases), high temperature up to 38°C (7 cases). Survey of 100 parents has shown that at least one adverse effect in 4 weeks period was noted only in 23 children: local reactions (induration, redness at the injection site) in 14 cases, general reactions (temperature increase up to 38.7°C) in 9 cases. No adverse effects needed medical intervention. Conclusion. Adverse effects were recorded in less than 1% of all cases according to local paediatricians appointments and in every 4th case according to parents questioning among all two years old children who undergone immunization with 4-valent meningococcal conjugate vaccine to A, C, W, Y serogroups.

Gauging the epidemic potential of a widely circulating non-invasive meningococcal strain in Africa.

Neisseria meningitidis colonizes the human oropharynx and transmits mainly via asymptomatic carriage. Actual outbreaks of meningococcal meningitis are comparatively rare and occur when susceptible populations are exposed to hypervirulent clones, genetically distinct from the main carriage isolates. However, carriage isolates can evolve into pathogens through a limited number of recombination events. The present study examines the potential for the sequence type (ST)-192, by far the dominant clone recovered in recent meningococcal carriage studies in sub-Saharan Africa, to evolve into a pathogen. We used whole-genome sequencing on a collection of 478 meningococcal isolates sampled from 1- to 29- year-old healthy individuals in Arba Minch, southern Ethiopia in 2014. The ST-192 clone was identified in nearly 60 % of the carriers. Using complementary short- and long-read techniques for whole-genome sequencing, we were able to completely resolve genomes and thereby identify genomic differences between the ST-192 carriage strain and known pathogenic clones with the highest possible resolution. We conclude that it is possible, but unlikely, that ST-192 could evolve into a significant pathogen, thus, becoming the major invasive meningococcus clone in the meningitis belt of Africa following upcoming mass vaccination with a polyvalent conjugate vaccine that targets the A, C, W, Y and X capsules.

Elimination of Epidemic Meningitis in the African Region: Progress and Challenges:2010-2016

Epidemics of meningococcal disease constitute a major public health challenge in Africa, affecting mostly the 24 countries of the meningitis belt. These epidemics led to a call for a call for a safe, effective and affordable conjugate vaccine against the major serogroup responsible for recent epidemics by leaders of the region. This paper documents experiences with efforts at eliminating epidemic meningitis in the African Region. The meningoccocal serogroup A conjugate vaccine was developed, licensed and offered to more than 235 million people through mass vaccination campaigns in 16 countries since 2010. Future plans include providing the vaccine to the remaining countries in the African Meningitis Belt and, to implement the vaccine into routine national infant immunization programme and to organise catch-up immunization campaigns every 5 years for unvaccinated <5 year-olds who had missed their routine vaccinations. The success of the project is evidenced by the large declines in cases of group A meningococcal disease since 2010, with no cases reported in vaccinated persons across the 16 countries, reflecting the highly effective nature of the vaccine. The successful control of serogroup A meningococcal disease has highlighted the need to tackle other meningococcal serogroups through development of polyvalent conjugate vaccines with the aim of eliminating epidemics of meningococcal meningitis in the African region.

True Versus False Bacteremia in Infants and Children Less Than 3 Years of Age.

The objective of this study was to examine patient history as well as clinical and laboratory features associated with true bacteremia versus false bacteremia in previously healthy febrile children ages 0 to 36 months in the era of polyvalent conjugate pneumococcal immunization. Using retrospective chart review, we examined history, physical examination, and laboratory characteristics associated with true and false bacteremia. We included subjects under 3 years old, with a positive blood culture obtained in the emergency department or clinic from July 2011 to July 2013, and fever defined as a temperature of greater than or equal to 100.4°F by history or examination. We excluded those with a previously known underlying disease process that could increase the risk for positive blood culture, for example, immunodeficiency, cancer, cystic fibrosis, or significant skin disorders such as severe eczema, as well as patients with any indwelling central line, shunt, or other implanted device, or recent hospitalization for a febrile illness. Thirty subjects (24%) had true bacteremia, and 97 (76%) had false bacteremia. In the multivariable logistic regression analysis, exposure to sick contacts (odds ratio, 0.1; 95% confidence interval, 0.01-0.6; P = 0.01) and increased hours to positive blood culture (odds ratio, 0.8; 95% confidence interval, 0.8-0.9; P < 0.001) remained significant factors associated with false bacteremia. Age, maximum reported temperature, and reported days of fever were not associated with true bacteremia. We found that previously healthy children with true bacteremia are more likely to grow bacteria faster on blood culture and lack exposure to sick contacts than children with false bacteremia.

Phylogenetic analysis of emergent Streptococcus pneumoniae serotype 22F causing invasive pneumococcal disease using whole genome sequencing.

Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13) in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD) has declined from 55% (n = 1492) in 2010 to 31% (n = 764) in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183) to 11% (n = 283). Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005–2015. Six phylogenetic lineages (n = 117) were identified among a serotype 22F/ST433 clonal complex (CC), including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events.

A phase II randomized, double-blind trial of a polyvalent vaccine-KLH conjugate (NSC 748933 IND# 14384) + OPT-821 versus OPT-821 in patients with epithelial ovarian (EOC), fallopian tube, or peritoneal cancer who are in second or third complete remission.

5517Background: Phase I data have demonstrated induction of antibody responses to a polyvalent vaccine conjugate (Globo-H, GM2, MUC1-TN, TF) with adjuvant OPT-821. We sought to determine if this co...

Synthesis, Characterization, and Immunological Properties of LPS-Based Vaccines Composed of O-Polysaccharides Conjugated with Recombinant Exoprotein A from &amp;lt;i&amp;gt;Pseudomonas aeruginosa&amp;lt;/i&amp;gt;

Pseudomonas aeruginosa remains one of the major pathogens affecting immunocompromised patients. LPS-based monovalent (MV) and polyvalent (PV) conjugate vaccines were prepared from the most prevalent strains of P. aeruginosa International Antigenic Typing Scheme (IATS) 6, 10, 11 and 20 to evaluate their immunogenicity and protective capacities from infection by the pathogens. Conjugation of the O-polysaccharide (O-PS) antigens of P. aeruginosa strains to the common immunogenic recombinant Exotoxin A (rEPA) supports the multi-antigenic approach for the development of a vaccine that provides cross protection against various strains of the pathogen. The O-PSs were indirectly conjugated through adipic acid dihydrazide (ADH) to rEPA by carbodiimidemediated condensation reaction. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL) or Freund's adjuvant compared with conjugates without adjuvant, unconjugated mixture of rEPA and O-PS emulsified with MPL, and sterile saline. The MV and PV vaccines emulsified with MPL adjuvant elicited the highest anti-O-PS IgM and IgG antibodies. Immunization of mice with MV vaccines derived from IATS 10, 11, and 20, emulsified with MPL adjuvant provided a high level of protection against the homologous bacterial strain. Similarly, high protection was obtained when mice were immunized using PV and challenged separately with bacterial strains 10, 11, and 20, but lower protection against the IATS 6 strain. Also, high cross protection of MV vaccine derived from O-PS of IATS 10 and 20 was obtained against P. aeruginosa IATS 11 strain. The in vivo protection correlated with the level of anti-O-PS IgG in the mice serum.

Synthesis of a conjugate of 3´-sialyllactoside with recombinant flagellin as a carrier protein and assessment of its immunological activity in comparison with that of a similar hemocyanin-based conjugate

In the framework of a project directed to the design of synthetic cancer vaccines based on carbohydrate chains of tumor-associated gangliosides, a new prototype cancer vaccine, a polyvalent carbohydrate-protein conjugate 1 with ∼11 3´-sialyllactoside ligands conjugated to one molecule of recombinant flagellin was synthesized. Dendritic cell vaccines were designed using the conjugate 1 and a neoglycoconjugate 2 consisting of ∼400 3´-sialyllactose residues linked to hemocyanin from a snail Megathura crenulata (KLH). Comparative ELISA study was conducted to assess the induction of carbohydrate-specific immunoglobulins IgM and IgG after immunization of mice with conjugates 1 and 2 and dendritic cells loaded with 1 and 2. Synthetic polyacrylamide carrier conjugated to 3´-sialyllactose ligands and B16F0 melanoma cells were used as coating antigens.

Antitumor effect of the conjugate of synthetic carbohydrate moiety of the tumor-associated ganglioside GM3 with hemocyanin in combination with the cytostatic cyclophosphamide

Based on the carbohydrate chains of tumor-associated ganglioside, polyvalent carbohydrate-protein conjugate 1 was synthesized suitable as a prototype of a cancer vaccine containing ∼400 residues of 3′-(N-acetyl-a-neuraminyl)lactose (3′-sialyllactose) per macromolecular complex of the hemocyanin protein of the Megathura crenulata keyhole limpet. The integrated antitumor effect of neoglycoconjugate 1 in combintion with the cytostatic cyclophosphamide was studied on the C57Bl/6 mice with transplanted melanoma B16/F0 cells.

Quantitative Proton Nuclear Magnetic Resonance evaluation and total assignment of the capsular polysaccharide Neisseria meningitidis serogroup X

Neisseria meningitidis constitutes the main cause of meningococcal disease in infants. Serogroups A, B, C, W135, Y, and X have the higher incidence in young children and teenagers. The use of polyvalent conjugate carbohydrate-based vaccines has decreased the meningococcal infection around the world. Recently, the serogroup X has been found to be responsible of different outbreaks of meningococcal diseases, mainly in "Meningitis Belt" of Africa and the structure of the repetitive unit of the capsular polysaccharide has been confirmed through a monodimensional (13)C NMR study. No further characterization studies have been carried out, especially with the use of other nuclei. In this paper a novel method for quantification of the N. meningitidis serogroup X by proton qNMR is reported. Deep characterization of the serogroup X polysaccharide was also carried out by combination of correlation experiments involving (13)C, (1)H, and (31)P nuclei.

SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections.

Synthesis, Characterization, and Thermodynamic Study of a Polyvalent Lytic Peptide−Polymer Conjugate As Novel Anticancer Agent

We designed and synthesized a new polyvalent lytic peptide-polymer conjugate as a novel chemotherapeutic agent capable of overcoming multidrug resistance. A hexapeptide (KWKWKW or (KW)₃) was designed and conjugated to dextran in multiple copies to afford a polyvalent conjugate. A robust synthesis procedure involving click chemistry and the detailed characterization of the conjugate were reported here. The conjugate Dex-(KW)₃ exhibited significantly enhanced anticancer potency in vitro by up to 500-fold compared to monomeric (KW)₃. The LC₅₀ value was comparable to that of conventional lytic peptides which have more than 20 residues. No hemolytic activity was shown by the conjugates up to 300 μM. Thermodynamic study indicated that the binding of conjugates was predominantly entropy-driven while the binding of free peptides was mainly enthalpy-driven, implying a deeper penetration of conjugate into the core of lipid bilayer. The binding affinity of conjugate to neutral membrane is much higher than that to free peptide (K(conj) ≈ 8822.9 M⁻¹, K(pep) ≈ 1884.7 M⁻¹). In binding to negatively charged membrane, the conjugate surpassed free peptides at high concentrations when the binding of free peptides became saturated. The higher binding capability, attributed to the high local concentration of peptides mounted on a polymer backbone, explains the superior anticancer activity of polyvalent Dex-(KW)₃.

Synthesis of 2-aminoethyl 3′-(N-glycolylneuraminyl)-β-actoside and neoglycoconjugates based thereon — a prototype of a cancer vaccine and artificial antigens for the control of immune response

The 2-aminoethyl β-glycoside of the trisaccharide 3′-(N-glycolylneuraminyl)lactose was synthesized in the framework of studies of approaches to the preparation of synthetic tumor vaccines based on carbohydrate chains of tumor-associated gangliosides and converted to the corresponding 2-4-maleimidobutanoylamino)ethyl glycoside. The reaction of the latter with thiolated hemocyanin from the keyhole limpet Megathura crenulata (KLH) gave a carbo-hydrate—protein conjugate, a tumor vaccine prototype containing about 250 trisaccharide ligands attached to a macromolecular KLH complex. A polyvalent conjugate with a polyacryl-amide support was synthesized to be used as a coating antigen in the control of specificity and efficiency of immune response by enzyme-linked immunosorbent assay (ELISA).

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30dpi. The IgG antibodies appeared at 40dpi in inbred mice, and at 50dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30dpi. Antibody levels increased until the end of the experiment (80dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.

Synthesis of 3-aminopropyl β-glycoside of sialyl-3′-lactose and derived neoglycoconjugates as a tumor vaccine prototype and artificial antigens for the control of immune response

Starting from the biotechnologically available trisaccharide sialyl-3′-lactose, representing the carbohydrate portion of the tumor-associated ganglioside GM3, the corresponding 3-aminopropyl β-glycoside (1) and 3-(4-maleimidobutanoylamino)propyl glycoside were synthesized. The reaction of the latter with a thiolated derivative of the Megathura crenulata hemocyanine (KLH) afforded a carbohydrate—protein conjugate, a tumor vaccine prototype containing about 330 trisaccharide ligands attached to KLH. N-Stearoylation of ligand 1 gave the model neoglycolipid for comparative study of the activity of mono-and polyvalent immunogens and the natural ganglioside GM3. A monovalent conjugate, in which ligand 1 is linked to biotin through an oligo(ethylene glycol) spacer and a polyvalent conjugate with a polyacrylamide carrier were also prepared. These conjugates are meant as covering antigens to assess the specificity and efficiency of the immune response in the ELISA assay.

Pneumococcal Vaccine for Infants and Toddlers

SUMMARYThe South African launch of Prevenar®, a new polyvalent conjugate vaccine, has resulted in many questions being posed by health care providers and parents alike. Without a government policy on the role of this vaccine and considering its high cost, there is much debate on its place in therapy, cost benefit and long term epidemiological effect. This article provides an overview of the vaccine, its known advantages, potential disadvantages, place in therapy and administration.

258 A phase I trial in patients with solid tumours using autologous dendritic cells loaded with mannan-conjugated recombinant MUC1 protein

Antigens such as ganglioside GD3, neutral glycolipid Lewisy (Ley) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3–KLH, Ley–KLH, MUC1–KLH and MUC2–KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.

Vaccination of Cystic Fibrosis Patients Against Pseudomonas aeruginosa Reduces the Proportion of Patients Infected and Delays Time to Infection

Cystic fibrosis (CF) almost always leads to chronic airway infection with Pseudomonas aeruginosa. Despite advances in antibiotic therapy, after chronic infection rapid deterioration in lung function occurs, increasing morbidity and mortality. Prevention of infection by vaccination is desirable, but earlier trials produced disappointing results. The promising short term immunogenicity and safety of a new P. aeruginosa vaccine prompted us to evaluate its long term efficacy. We conducted a 10-year retrospective analysis of outcomes in a group of vaccinated patients. In 1989-1990, 30 young children with CF, mean age 7 years, with no prior history of infection with P. aeruginosa, were vaccinated against P. aeruginosa with a polyvalent conjugate vaccine. We report the follow-up of 26 of these patients from 1989 to 2001. The patients were given yearly vaccine boosters. Comparisons were made with a CF patient control group matched for gender, age and, where possible, genetic mutation. Vaccinated patients and controls were attending a single CF clinic and received the same clinical management throughout the study period. Main outcomes were time to infection, proportion of patients infected, development of P. aeruginosa mucoid phenotype, lung function and body weight. The time to infection with P. aeruginosa was longer in the vaccination group than in the control group, and fewer vaccinated patients than controls became chronically infected (32% versus 72%; P < 0.001). The proportion of mucoid infections was higher in the control group (44%) than in the vaccinated group (25%). Patients >/=18 years of age at the end of the study had a lower mean forced expiratory volume at 1 s (FEV1) than did those 13-17 years of age, but this difference was small in the vaccinated group (73.6% versus 83.7%) compared with the controls (48.0% versus 78.7%). In the >/=18 year age category the mean FEV1% at 10 years was 73.6% (vaccinated) and 48.0% (controls) (P < 0.05). In the vaccinated group only 11 (44%) of 25 patients were underweight at the 10-year follow-up compared with 18 (72%) of 25 at the beginning of the study. In the control group 17 (68%) of 25 patients were underweight at 10-year follow-up compared with 16 (64%) of 25 at the beginning of the study. Regular vaccination of young CF patients for a period of 10 years with a polyvalent conjugate vaccine reduced the frequency of chronic infection with P. aeruginosa. This was associated with better preservation of lung function. Vaccinated patients gained more weight during the study period, a possible indication of an improved overall health status.

Quality Control of Polyvalent Pneumococcal Polysaccharide-Protein Conjugate Vaccine by Nephelometry

A nephelometric method was used for quantitative analysis of individual polysaccharides (PSs) in a polyvalent pneumococcal conjugate vaccine using CRM197 as carrier protein. Using this method, the individual types 4, 6B, 9V, 14, 18C, 19F and 23F PSs were found to range between 82·3 to 119% of the manufacturer's indicated values.During conjugation using reductive amination, pneumococcal PS was first oxidized to introduce aldehyde groups. Higher or lower levels of antigen-antibody reaction were observed in periodate activated and then reduced PS of some serotypes compared to non-treated PS. Use of oxidized and reduced PS may provide an early indication of change in conjugation process. Furthermore, since the final monovalent and polyvalent conjugate vaccines gradually change during the storage period, the nephelometry provides an useful analytical method for stability study of these vaccines. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

Comparison of antibody titers after immunization with monovalent or tetravalent KLH conjugate vaccines

Antigens such as ganglioside GD3, neutral glycolipid Lewis y (Le y) and mucins MUC1 and MUC2 are over-expressed on the cell surface of many tumors. We have shown previously that conjugation of antigens such as these to keyhole limpet hemocyanin (KLH) and the use of immunological adjuvant QS-21 is the optimal approach for inducing high titer IgM and IgG antibodies. These antibodies are able to bind with natural antigens on the tumor cell surface and mediate complement dependent cytotoxicity and/or antibody dependent cell mediated cytotoxicity. Immunization of patients with monovalent vaccines containing these and a variety of other antigens have demonstrated both the consistent immunogenicity and the safety of these vaccines. Now, in preparation for the use of polyvalent conjugate vaccines in the clinic, we have addressed for the first time with conjugate vaccines against cancer antigens several questions in the pre-clinical setting, including whether immunogenicity of the individual components is decreased in the polyvalent vaccine and issues relating to vaccine formulation and administration. We have immunized groups of mice with GD3–KLH, Le y–KLH, MUC1–KLH and MUC2–KLH conjugates and QS-21 separately or mixed and administered at one or four sites. High titer IgM and IgG antibodies were induced against each of the four antigens whether administered singly in separate mice, at separate sites in the same mice, or mixed and administered at a single site or at four sites, or administered subcutaneously (s.c.) or intraperitoneally (i.p.). These antibodies reacted specifically with the respective antigens and tumor cells expressing these antigens. There was no evidence of suppression of the antibody response against any one of the antigens by the presence of the other conjugates in the vaccine. Immunogenicity of the four individual antigens conjugated to KLH and QS-21 is not affected by mixing the four together and administering them at a single subcutaneous site.